Compositions and methods for treating toxoplasmosis, cryptosporidiosis, and other apicomplexan protozoan related diseases

ABSTRACT

Compositions and methods for the treatment of toxoplasmosis, caused by the infectious eukaryotic parasite  Toxoplasma gondii  ( T. gondii ) and for the treatment of cryptosporidiosis, caused by the infectious eukaryotic parasites  Cryptosporidium parvum  ( C. parvum ) and  Cryptosporidium hominus  ( C. hominus ) are described. In particular, the present disclosure is directed to compositions and methods for inhibiting either  T. gondii  calcium dependent protein kinases (TgCDPKs) or  C. parvum  and  C. hominus  calcium dependent protein kinases (CpCDPKs) using pyrazolopyrimidine and/or imidazo[1,5-a]pyrazine inhibitors, of the formula, 
                         
wherein the variables X, Y, Z, L, R 1 , and R 3  are defined herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is divisional application of U.S. application Ser. No.13/561,896, filed Jul. 30, 2012, which is a continuation-in-part of theInternational Application Serial No. PCT/US11/23047, filed on Jan. 28,2011, which claims the benefit of the filing dates of U.S. ProvisionalPatent Application Ser. No. 61/299,286, filed Jan. 28, 2010, and U.S.Provisional Patent Application Ser. No. 61/358,045, filed Jun. 24, 2010,and also claims the benefit of the filing date of U.S. ProvisionalPatent Application Ser. No. 61/534,285 filed Sep. 13, 2011, each ofwhich are hereby incorporated by reference in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with U.S. government support under P01 AI067921,R01AI080625, R01AI50506, R01 AI089441, and R01 GM086858 awarded by theNational Institutes of Health (NIAID). The U.S. Government has certainrights in the invention.

FIELD OF THE INVENTION

The present disclosure is generally directed to compositions and methodsfor treating apicomplexan protozoan related disease, such astoxoplasmosis and cryptosporidiosis.

BACKGROUND OF THE INVENTION

The apicomplexan protozoans Cryptosporidium parvum and Toxoplasma gondiiare ubiquitous parasites that infect humans and domesticated animals.Recently C. hominus was recognized to be distinct from C. parvum, anddoes not appear to infect domesticated animals, but rather appearslimited to human infections. C. parvum and C. hominus are infectiousparasites of major health concern in humans as they are a common causeof illness transmitted by water. (See White A C. Chapter 280:Cryptosporidiosis (Cryptosporidium hominis, Cryptosporidium parvum, andOther Species) in Mandell, Bennett, & Dolin: Principles and Practice ofInfectious Diseases, 6th ed. Publ: Churchill Livingston (2005)) C.parvum and C. hominus infections result in debilitating diarrhea thatcan be life-threatening in immunocompromised patients.

Recent studies have implicated Cryptosporidium spp. in around 15-20% ofchildhood diarrheal disease in the developing world. (See Samie et al.,Cryptosporidium species: preliminary descriptions of the prevalence andgenotype distribution among school children and hospital patients in theVenda region, Limpopo Province, South Africa. Exp. Parasitol. 114,314-322 (2006); and Ajjampur et al., Closing the diarrhea diagnostic gapin Indian children by the application of molecular techniques. J. Med.Microbiol. 57, 1364-1368 (2008)) Currently, nitazoxanide is the onlyapproved therapy for cryptosporidiosis but it is expensive and has notbeen shown to be effective in treating immunocompromised hosts. T.gondii may be the most common infectious eukaryotic parasite in humans,based on serosurveys. (See Montoya et al., Chapter 276: Toxoplasmagondii in Mandell, Bennett, & Dolin: Principles and Practice ofInfectious Diseases, 6th ed. Publ: Churchill Livingston (2005))Transmitted primarily through undercooked meat or accidental ingestionof cat feces, T. gondii infection presents major health concerns inimmunocompromised hosts, where it causes toxoplasmic encephalitis, andin pregnancy, where it can result in severe birth defects ormiscarriage. Sulfadiazine and pyrimethamine are the current therapiesfor toxoplasmosis, but they can cause nephrotoxicity, rash, andadditional complications in pregnancy. Thus, new therapies for treatinginfections caused by both parasites are greatly needed.

In T. gondii, calcium-regulated signaling is associated with a number ofcellular functions such as secretion, gliding motility and host cellinvasion. (See Nagamune and Sibley, Comparative genomic and phylogeneticanalyses of calcium ATPases and calcium-regulated proteins in theapicomplexa. Mol. Biol. Evol. 23, 1613-1627 (2006); and Billker et al.,Cell Host Microbe. 2009 Jun. 18; 5(6):612-22. Calcium-dependentsignaling and kinases in apicomplexan parasites) The proper control ofintracellular calcium levels is important for host cell invasion and T.gondii use several mechanisms for the uptake and release of calcium.Furthermore, this organism contains specialized calcium-regulatedsignaling enzymes, including a unique family of calcium-dependentprotein kinases (CDPKs) which are present in plants, ciliates and greenalgae but not in animals. (See Doerig et al., Protein kinases as targetsfor antimalarial intervention: kinomics, structure-based design,transmission-blockade, and targeting host cell enzymes. Biophysica etBiochimica Acta—Proteins and Proteomics 1754, 132-150 (2005)) Thesekinases are believed to be mediators of secretion, invasion, and glidingmotility. (See Nagamune and Sibley L D, supra; Billker et al., supra;and Kieschnick et al., C. Toxoplasma gondii attachment to host cells isregulated by a calmodulin-like domain protein kinase. J. Biol. Chem.276, 12369-12377 (2001)) T. gondii, C. parvum, and C. hominus are highlyrelated obligate intracellular parasites. While much less is known aboutthe role of calcium signaling in C. parvum and C. hominus, it appearsthat many calcium-regulated signaling processes are conserved from T.gondii to C. parvum. (See Chen et al., Apical Organelle discharge byCryptosporidium parvum is temperature, cytoskeleton, and intracellularcalcium dependent and required for host cell invasion. Infect. Immun.72, 6806-16 (2004)) C. parvum and C. hominus also possess CDPKs that arebelieved to play important roles in calcium-regulated processes and theyare virtually identical in these two spp. Thus inhibitors of C. parvumCDPKs would be expected to inhibit C. hominus CDPK.

The roles that CDPKs play in calcium signaling in T. gondii, C. parvumand C. hominus make this family of kinases intriguing targets for thedevelopment of anti-parasitic agents.

SUMMARY OF THE INVENTION

The present disclosure is generally directed to compositions and methodsfor the treatment of apicomplexan-related disorders, including but notlimited to toxoplasmosis, caused by the infectious eukaryotic parasiteToxoplasma gondii (T. gondii), cryptosporidiosis, caused by theinfectious eukaryotic parasites Cryptosporidium parvum (C. parvum) andCryptosporidium hominus (C. hominus), and malaria, caused by theeukaryotic parasites Plasmodium falciparum (P. falciparum) andPlasmodium berghei (P. berghei). In one embodiment, the presentdisclosure is directed to compositions and methods for inhibitingapicomplexan calcium dependent protein kinases, including but notlimited to T. gondii calcium dependent protein kinases (TgCDPKs), C.parvum and C. hominus calcium dependent protein kinases (CpCDPKs), or P.falciparum and P. berghei calcium dependent protein kinase 4 (PfCDPKs)using pyrazolopyrimidine inhibitors, or in another embodiment,Imidazo[1,5-a]pyrazine inhibitors, both classes of compounds designed tobe inactive against mammalian kinases.

In one aspect, the present disclosure provides compounds of the formula(I),

and pharmaceutically acceptable salts thereof, wherein R¹, R³, X, Y, andZ are defined herein.

In another aspect, the present disclosure provides methods for treatingan apicomplexan protozoan related disease comprising providing to apatient in need of such treatment a therapeutically effective amount ofeither (i) a bumped kinase inhibitor or (ii) a pharmaceuticalcomposition comprising a bumped kinase inhibitor and a pharmaceuticallyacceptable excipient, carrier, or diluent.

In another aspect, the present disclosure provides methods for treatingan apicomplexan protozoan related disease comprising providing to apatient in need of such treatment a therapeutically effective amount ofeither (i) a bumped kinase inhibitor or (ii) a pharmaceuticalcomposition comprising a bumped kinase inhibitor and a pharmaceuticallyacceptable excipient, carrier, or diluent.

In another aspect, the present disclosure provides methods for treatingan apicomplexan protozoan related disease comprising providing to apatient in need of such treatment a therapeutically effective amount ofeither a compound of formula (II) or (ii) a pharmaceutical compositioncomprising a compound of formula (II) and a pharmaceutically acceptableexcipient, carrier, or diluent, wherein the compound of formula (II) is

wherein R¹, R³, L, X, Y, and Z are defined herein.

One aspect of the present disclosure provides a method of treatingmalaria comprising administering to a subject an effective amount of anyone of compounds of the disclosure that inhibits the activity of P.falciparum and P. berghei calcium dependent protein kinase 4 (PfCDPK4).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of calcium-dependence of TgCDPK1activity. Enzyme activity was measured at a constant concentration of0.5 mM EGTA with increasing concentrations of CaCl₂ using the luciferasebased assay format. The precise quantity of free Ca²⁺ was determined bymeasuring fluorescence intensity of Fluo-3 and Calcium green dyes(Invitrogen Corp. Carlsbad, USA) in the reaction buffer at excitationand emission wave lengths of 506 and 526 nM (Fluo-3) and 503 and 532 nM(Calcium green). No activity was observed with equimolar concentrationsof EGTA and CaCl₂ (0 free Ca²⁺) but significant kinase activity wasobserved by gradually increasing the CaCl₂ concentration in the mixture.The reaction reached maximal activity at approximately 18 μM free CaCl₂.

FIGS. 2a-2e are graphical representations of the crystal structure ofcalcium-free TgCDPK1. FIG. 2a : Association of the kinase domain (green)and the calmodulin-like calcium regulatory domain (orange) buriesapproximately 1400 A2 of accessible surface area per domain. Thejunction domain, connecting the kinase and regulatory domains, is shownin brown. This interaction surface between kinase and regulatory domainsis entirely lost during the transition to the Ca²⁺-bound form in whichthe regulatory domain relocates to lie along the opposite surface of thekinase domain [Wernimont et al. submitted for publication, PDB accessionnumber 3hx4], the left side in the orientation shown. The active site isshown with bound inhibitor, NA-PP2 (magenta). FIG. 2b : Comparativeanalysis of the ATP binding pocket of kinases reveals the presence of aglycine in the position of the gatekeeper residue (bold) in TgCDPK1 anda C. parvum CDPK1 ortholog. Medium-sized side chains, threonine andserine, are found at this position in other apicomplexan CDPKs, E.tenella (EtCDPK1) and P. falciparum (PfCDPK1 and PfCDPK4). Animal CDPKstend to have large gatekeeper residues such as phenylalanine. FIG. 2cand FIG. 2d : Stereo view of the active site with the bumped kinaseinhibitors NA-PP2 (2 c) and NM-PP1 (2 d) bound. The orientation is as inpanel 1 a. Residues within 6 Å of the inhibitor are shown as sticks. Theunusual glycine gatekeeper residue that permits inhibition by bumpedkinase inhibitors is highlighted in cyan. Difference electron density,contoured at +3.5σ (green mesh) and −3.5σ (pink mesh), was calculatedafter omission of the inhibitor. FIG. 2e : TgCDPK1 active site in thevicinity of the gatekeeper residue. The surface of TgCDPK1 is shown inwhite and is slabbed through to show the adenine pocket of theATPbinding site. The glycine at the gatekeeper position of TgCDPK1enlarges the active site pocket and facilitates binding of bumped kinaseinhibitors (NA-PP2 is magenta sticks and NM-PP1 is violet sticks).Larger amino acids, such as Met shown here (brown sticks and protrudingsurface) from superposition of a C. parvum ortholog (PDB ID 3hko; boundAMP-PNP shown as brown sticks) are more typical at this gatekeeperposition, and prevent binding of compounds with large, hydrophobic“bumps” on the adenine ring. This is apparent by the brown surfaceprotrusion of the Met clearly clashing with the “bump” of each of theBKIs in our crystal structures.

FIG. 3 is a graphical representation of the nuclear and cytosoliclocalization of TgCDPK1 in T. gondii. Live T. gondii cells transientlytransfected with wild type TgCDPK1-GFP were fixed in 4%paraformaldehyde. Samples were probed with anti-GFP followed byanti-rabbit Ig coupled to Texas red, stained with DAPI and viewed. GFP,green; DAPI, blue.

FIGS. 4a-4b are graphical representations demonstrating the effects ofbumped kinase inhibitors on T. gondii binding to and invasion ofmammalian cells. For these assays, T. gondii expressing β-galactosidase(10³ per well) were allowed to infect fibroblasts in a 96 well plate.For “invasion” assays (FIG. 4a ), compounds were added to the wellsbefore infection with T. gondii. For “growth” assays (FIG. 4b ),compounds were added to the indicated final concentration, four hoursafter infection with T. gondii cells. After 20 hours, the cells werelysed and β-galactosidase activity was assessed by adding 0.5 mMchlorophenol red-β-d-galactoside (CPRG), incubating at 37° C., and thenmeasuring absorbance at 595 nm.

FIGS. 5a-5b are graphical representations demonstrating TgCDPKgatekeeper mutant reduction of sensitivity to BKIs. T. gondii clonallines expressing HA-tagged versions of TgCDPK1 or a G128M mutant alongwith GFP and β-galactosidase were generated. Western blot (FIG. 5a ) ofprotein separated on a 10% acrylamide gel and probed with anti-HA.Anti-GFP served as a loading control. Invasion assays were performedwith increasing concentrations of BKIs NA-PP2 (FIG. 5b ) using theparental cell line and the transfectants additionally expressing wildtype TgCDPK1 and TgCDPK1(G128M). Assays were done in triplicate and meanand standard deviations normalized to the no compound (vehicle alone)control are presented.

FIG. 6 is a K_(m) plot of ATP for TgCDPK1 (2 nM) using the SPA assay tomeasure phosphorylation of a biotinylated peptide substrate by labeledγ-phosphate of ATP after 90 minutes incubation at 30° C.

FIG. 7 is a K_(m) plot of Syntide 2 substrate for TgCDPK1 (2 nM) usingthe SPA assay to measure phosphorylation of a biotinylated peptidesubstrate by labeled γ-phosphate of ATP after 90 minutes incubation at30° C.

FIG. 8 is a K_(m) plot of ATP using the luciferase based KinaseGlo assaythat measures consumption of ATP after TgCDPK1 (2 nM) phosphorylation ofpeptide substrate Syntide 2 at 30° C. during a 90 minute reaction time.Remaining ATP in the reaction was measured after addition of KinaseGloreagent by luminescence light production. Omitting peptide substrate orenzyme led to virtually no consumption of ATP (not shown).

FIG. 9 is a K_(m) plot of Syntide 2 substrate using the luciferase basedKinaseGlo assay as described for FIG. 8.

FIG. 10a is a representative image from the untreated CDPK-GFP dataset.Intracellular parasites are marked by asterisks.

FIG. 10b shows the change in the number of intracellular parasites upondrug treatment. The number of intracellular parasites was determined bysubtracting the number of extracellular parasites (SAG+) by the numberof total parasites (GFP+) and expressed as a ratio to the number of hostcells observed (as measured by DAPIstained nuclei). Each sample wasnormalized to the untreated control of the same line.

FIG. 10c shows the data used to derive the graph in FIG. 8(b). Note thatcells expressing the gatekeeper mutant CDPK1(G128M) are less sensitiveto the drug. The drug additionally appears to affect adhesion.

FIG. 11a is a representative set of images of CDPK1(G128M)-GFP mixedwith wild type, in the absence of drug. Vacuoles containing multipleGFP-expressing parasites are marked with * and wild type vacuoles aremarked with

.

FIG. 11b shows the number of vacuoles with wild type (GFP−) ortransfected parasites (GFP+), per host cell nucleus, in the absence andpresence of drug for the three cell mixtures.

FIG. 11c shows the data used to derive the graphs. Note that theparasite line overexpressing CDPK1(G128M)-GFP was resistant to theeffects of 1 μM NA-PP2, as compared to the co-cultured wild typecontrol.

FIG. 12. (A) TgCDPK1 and CpCDPK1 inhibitors 1-3. IC₅₀ values shown arethe average of three assays±(SEM). (B) C. parvum parasite numbers (Yaxis) present after 24 hours of infection of human intestinal cells(HCT-8) with C. parvum sporozoites (1:1 ratio HCT-8 cells: C. parvumsporozoites) in the presence of varying concentrations of inhibitors1-3. (left graph) Cultures were exposed to each inhibitor at the time ofinfection. (right graph) Cultures were exposed to each inhibitor 1 hourafter infection.

FIG. 13 is a graphical representation of the crystal structure of (leftpanel) Superposition of the active sites of TgCDPK1.3 (green sticks andlight green ball-and-sticks) and CpCDPK1.3 (orange sticks andball-and-sticks) complexes (Pdb entries 3i7b and 3ncg). (right panel)Overlay of the TgCDPK1.5h and CpCDPK1.5h complexes (Pdb entries 3n51 and3mwu). Only residues within 6 Å of the bound inhibitors are shown andthe unique glycine gatekeeper residue is shown as ball-and-sticks.

FIG. 14 shows in vitro activities of previously-described kinaseinhibitors against TgCDPK1. Values shown are the average of threeassays.

FIG. 15 shows the structure of calcium-free, inactive CDPK1 versuscalcium bound, activated CDPK1. The kinase domains of calcium-freeTgCDPK1 (green cartoon) and calcium-bound CpCDPK1 (orange cartoon) aresuperimposed to highlight the dramatic conformational change that occursin the CDPK activating domain (CAD) upon calcium activation. In theabsence of calcium, access to the active site (marked by boundinhibitors shown as ball-and-sticks) by protein substrates is occludedby the CAD (cyan cartoon). Upon binding calcium (gray spheres), the CAD(beige cartoon) is repositioned to the opposite surface of the kinasedomain so that protein substrates may now bind. Despite this largeconformational rearrangement of the CAD, the active site of the kinasedomain in the vicinity of the bound inhibitors remains largelyunchanged.

FIG. 16 shows activities of previously-described kinase inhibitors inthe immunosuppressed mouse C. parvum model. SCID-beige C57BL6 mice wereinfected with 106 C. parvum oocysts on day 0. They were treated withonce a day oral gavage with compound 29 (50 mg/kg/day, N=3), compound150 (50 mg/kg/day, N=4), or Paromomycin (2 g/kg/day, N=3) on days 3-7and 10-14. Infected controls (N=4) were gavaged with vehicle (3%tween80/7% EtOH/90% saline) alone. Shown are mean of the parasitenumbers detected in 25 mg of stool from each mouse, by qRT-PCR. At theend of treatment, parasites were not detected in the drug treatedgroups, but the parasite burden quickly rebounded in the Paromomycingroup, slowly with 29, and not with 150.

FIGS. 17a-17d are graphical representations demonstrating the blockingeffects of Compound 29 on exflagellation of P. berghei and P.falciparum. For exflagellation blocking activities of Compound 29 inmouse blood (FIG. 17a ), mice were infected with P. berghei expressingPfCDPK4-2x-myc, and treated i.p. with 50 mg/kg Compound 29 or NA-PP2 orvehicle control. Plots show the average number of exflagellation eventsper high powered field (error bars are standard error of the mean) andmouse blood Compound 29 or NA-PP2 concentration per time point. Therewas complete suppression of male gametocyte differentiation toexflagellating units for 14 hours in the presence of Compound 29relative to NA-PP2 treated or untreated controls. Data arerepresentative of three experiments. For in vitro studies (FIG. 17b ),Compound 29's effects on exflagellation of P. berghei WT and P. bergheiexpressing PbCDPK4 or PfCDPK4 were studied. Exflagellation inducingmedium containing different concentrations of Compound 29 or vehicle wasmixed with gametocytes from wild type or cdpk4 complemented mutants invitro. Exflagellation centers in 10 microscopic fields are expressed asa percentage of a solvent control. Error bars show standard deviationsof triplicate measurements from 2 infected mice per parasite strain. Forstudies of the effect of treating mice with Compound 29 on P. bergheitransmission to A. stephensi (FIG. 17c ), mice were infected with P.berghei expressing GFP and treated with vehicle, 3 mg/kg, or 10 mg/kgCompound 29 or NA-PP2. The infectivity of mice was calculated as thegeometric mean oocyst number on 8-20 dissected mosquito midguts. Eachbar shows the arithmetic mean oocyst number generated by 3 infectedmice. Error bars show standard deviations between mouse infections.Fluorescence micrographs illustrate typical infection levels inmosquitoes fed on control and Compound 29 treated mice. Scale bar=500μM. Data are representative of three experiments. For studies of theeffect of Compound 29 on P. falciparum exflagellation and infection ofmosquitos (FIG. 17d ), blood containing P. falciparum NF-54 straingametocytes and 3 μM, 1 μM 0.3, 0.1, or 0.05 μM final concentration ofCompound 29 or 3, 0.3 or 0.05 μM final concentration of NA-PP2, or nocompound controls was fed to A. stephensi. A complete suppression ofexflagellation was observed with 1 and 3 μM Compound 29. Blockingexflagellation with Compound 29 correlated well with the prevention ofoocyst and infective sporozoites formation. Sexual stage development inmosquitoes fed with 0.1 μM was not completely but significantly reducedas shown by >86% reduction in the number of oocyst and infectivesporozoites. In a repeat experiment, exflagellation events in thepresence of Compound 29 were suppressed completely at 1 or 3 μM,and >90% at 0.3 μM.

DETAILED DESCRIPTION OF THE INVENTION

The following description provides specific details for a thoroughunderstanding of, and enabling description for, embodiments of thedisclosure. However, one skilled in the art will understand that thedisclosure may be practiced without these details. In other instances,well-known structures and functions have not been shown or described indetail to avoid unnecessarily obscuring the description of theembodiments of the disclosure.

In one aspect, the present disclosure provides compounds of the formula(I),

or a pharmaceutically acceptable salt thereof, wherein

-   X, Y, and Z are defined by either: (i) X is N, Y is C, and Z is N;    or (ii) X is C, Y is N, and Z is C(H);-   R¹ is C₂₋₆ alkyl, C₁₋₆ haloalkyl, —C₁₋₄ alkyl-R¹², C₂₋₆ alkenyl,    C₂₋₆ alkynyl, C_(3-s) cycloalkyl, monocyclic heterocyclyl,    monocyclic heteroaryl, or phenyl, wherein    -   the cycloalkyl, heterocyclyl, heteroaryl, and phenyl groups are        each optionally substituted with one or two R¹¹ groups;    -   each R¹¹ is independently C₁₋₆ alkyl, C₁₋₆ haloalkyl, —C(O)R,        —C(O)OR, —C(O)NR₂, —S(O)₂NR₂, or —S(O)₂R;    -   and    -   R¹² is —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,        —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR,        —N(R)C(O)NR₂, phenyl, monocyclic heteroaryl, C₃₋₈ cycloalkyl, or        monocyclic heterocyclyl, wherein the aryl, heteroaryl, C₃₋₈        cycloalkyl, and heterocyclyl groups are each optionally        substituted by one, two, or three groups that are each        independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,        —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R,        —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂;-   R³ is one of the formulas,

wherein

-   -   n is 0, 1, or 2;    -   Q is —O—, —S—, or —N(R^(Q))—, wherein R^(Q) is hydrogen or C₁₋₆        alkyl; and    -   R³³ is C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆ haloalkyl, C₃₋₈        cycloalkyl, heterocyclyl, aryl, arylC₁₋₆ alkyl, heteroaryl, or        heteroarylC₁₋₆ alkyl wherein the alkyl, aryl, arylalkyl,        heteroaryl, and heteroarylalkyl are optionally substituted with        one, two, three, or four groups that are each independently        halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰,        —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰,        —OC(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰,        —N(R²⁰)C(O)OR²⁰, or —N(R²⁰)C(O)N(R²⁰)₂, wherein each R²⁰ is        independently hydrogen or C₁₋₆ alkyl.    -   each R³² is independently halogen, cyano, nitro, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, —OR³⁴, —SR³⁴, —N(R³⁴)₂, —C(O)R³⁴, —C(O)OR³⁴,        —C(O)N(R³⁴)₂, —S(O)₂R³⁴, —OC(O)R³⁴, —OC(O)OR³⁴, —OC(O)N(R³⁴)₂,        —N(R³⁴)C(O)R³⁴, —N(R³⁴)C(O) OR³⁴, or —N(R³⁴)C(O)N(R³⁴)₂, wherein        each R³⁴ is independently hydrogen or C₁₋₆ alkyl;    -   and    -   R³⁵ is hydrogen or C₁₋₆ alkyl;

-   and

-   each R is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆    haloalkyl, C₃₋₈ cycloalkyl, heterocyclyl, aryl, arylC₁₋₆ alkyl,    heteroaryl, or heteroarylC₁₋₆ alkyl wherein the alkyl, aryl,    arylalkyl, heteroaryl, and heteroarylalkyl are optionally    substituted with one, two, three, or four groups that are each    independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,    —OR⁰, —SR⁰, —N(R⁰)₂, —C(O)R⁰, —C(O)OR⁰, —C(O)N(R⁰)₂, —S(O)₂R,    —OC(O)R⁰, —OC(O)OR⁰, —OC(O)N(R⁰)₂, —N(R⁰)C(O)R, —N(R⁰)C(O)OR⁰, or    —N(R⁰)C(O)N(R⁰)₂, wherein each R⁰ is independently hydrogen or C₁₋₆    alkyl,

-   provided that the compound is not

The disclosure further comprises subgenera of formula (I) in which thesubstituents are selected as any and all combinations of one or more ofstructural formula (I), n, Q, R¹, R³, R³², and R³³ as defined herein,including without limitation, the following:

Structural Formula I is One of Formulae (Ia)-(Ib):

R¹ is Selected from One of the Following Groups (1a)-(1ii):

-   (1a) R¹ is C₂₋₄ alkyl, —C₁₋₄ alkyl-R¹², C₂₋₄ alkynyl, C₃₋₈    cycloalkyl, monocyclic heterocyclyl, monocyclic heteroaryl, or    phenyl, wherein the cycloalkyl, heterocyclyl, heteroaryl, and phenyl    groups are each optionally substituted with one or two R¹¹ groups.-   (1b) R¹ is C₃₋₈ cycloalkyl, monocyclic heterocyclyl, monocyclic    heteroaryl, or phenyl, wherein the cycloalkyl, heterocyclyl,    heteroaryl, and phenyl groups are each optionally substituted with    one or two R¹¹ groups.-   (1c) R¹ is C₃₋₈ cycloalkyl; or a monocyclic heterocyclyl optionally    substituted with one R¹¹ group.-   (1d) R¹ is C₃₋₈ cycloalkyl.-   (1e) R¹ is monocyclic heterocyclyl optionally substituted with one    R¹¹ group.-   (1f) R¹ is piperidinyl or tetrahydropyranyl, each optionally    substituted with one R¹¹ group.-   (1g) R¹ is phenyl optionally substituted with one or two R¹¹ groups.-   (1h) R¹ is C₂₋₆ alkyl.-   (1i) R¹ is C₂₋₄ alkyl.-   (1j) R¹ is isopropyl or t-butyl.-   (1k) R¹ is t-butyl.-   (1l) R¹ is isopropyl.-   (1m) R¹ is C₂₋₆ alkyl or —C₁₋₄ alkyl-R¹².-   (1n) R¹ is —C₁₋₄ alkyl-R¹².-   (1o) R¹ is —C₁₋₂ alkyl-R¹².-   (1p) R¹ is —CH₂—R¹²-   (1q) Any one of groups (1m)-(1p), wherein R¹² is —C(O)OR, —C(O)NR₂,    phenyl, monocyclic heteroaryl, C₃₋₈ cycloalkyl, or monocyclic    heterocyclyl, wherein the phenyl, heteroaryl, C₃₋₈ cycloalkyl, and    heterocyclyl groups are each optionally substituted by one, two, or    three groups that are each independently halogen, cyano, nitro, C₁₋₆    alkyl, C₁₋₆ haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂,    —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or    —N(R)C(O)NR₂;-   (1r) Any one of groups (1m)-(1p), R¹² is —C(O)OR or —C(O)NR₂.-   (1s) Any one of groups (1m)-(1p), R¹² is phenyl, monocyclic    heteroaryl, C₃₋₈ cycloalkyl, or monocyclic heterocyclyl, each    optionally substituted by one, two, or three groups that are each    independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,    —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R,    —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂.-   (1t) Any one of groups (1m)-(1p), wherein R¹² is phenyl, monocyclic    heteroaryl, C₃₋₈ cycloalkyl, or monocyclic heterocyclyl, wherein the    phenyl, heteroaryl, C₃₋₈ cycloalkyl, and heterocyclyl groups are    each optionally substituted by one or two groups that are each    independently halogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂,    —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or    —N(R)C(O)NR₂.-   (1u) Any one of groups (1m)-(1p), R¹² is phenyl or monocyclic    heterocyclyl, each optionally substituted by one, two, or three    groups that are each independently halogen, cyano, nitro, C₁₋₆    alkyl, C₁₋₆ haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂,    —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or    —N(R)C(O)NR₂.-   (1v) Any one of groups (1m)-(1p), R¹² is monocyclic heterocyclyl    optionally substituted by one, two, or three groups that are each    independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,    —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R,    —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂.-   (1w) Any one of groups (1m)-(1p), wherein R¹² is monocyclic    heterocyclyl optionally substituted by one or two groups that are    each independently halogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂,    —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or    —N(R)C(O)NR₂.-   (1x) Any one of groups (1m)-(1p), R¹² is piperidinyl or    tetrahydropyranyl, each optionally substituted by one or two groups    that are each independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆    haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,    —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or    —N(R)C(O)NR₂.-   (1y) Any one of groups (1m)-(1p), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,    —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R,    —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂.-   (1z) Any one of groups (1m)-(1p), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A), —C(O)N(R^(A))₂,    —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,    —N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,    wherein each R^(A) is independently hydrogen, C₁₋₆ alkyl, C₂₋₆    alkenyl, C₁₋₆ haloalkyl, C₃₋₈ cycloalkyl, heterocyclyl, aryl,    arylC₁₋₆ alkyl, heteroaryl, or heteroarylC₁₋₆ alkyl.-   (1aa) Any one of groups (1m)-(1p), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,    —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or    —N(R)C(O)NR₂.-   (1bb) Any one of groups (1m)-(1p), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A), —C(O)N(R^(A))₂,    —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,    —N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,    wherein each R^(A) is independently hydrogen or C₁₋₆ alkyl.-   (1cc) Any one of groups (1m)-(1p), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, or —S(O)₂R.-   (1dd) Any one of groups (1m)-(1p), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R^(A), or —S(O)₂R^(A), wherein each    R^(A) is independently hydrogen or C₁₋₆ alkyl.-   (1ee) Any one of groups (1m)-(1p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂,—S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or—N(R)C(O)NR₂.

-   (1ff) Any one of groups (1m)-(1p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A),—C(O)N(R^(A))₂, —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,—N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,wherein each R^(A) is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₁₋₆ haloalkyl, C₃₋₈ cycloalkyl, heterocyclyl, aryl, arylC₁₋₆ alkyl,heteroaryl, or heteroarylC₁₋₆ alkyl.

-   (1gg) Any one of groups (1m)-(1p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A),—C(O)N(R^(A))₂, —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,—N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,wherein each R^(A) is independently hydrogen or C₁₋₆ alkyl.

-   (1hh) Any one of groups (1m)-(1p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, or—S(O)₂R.

-   (1ii) Any one of groups (1m)-(1p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), or —S(O)₂R^(A),wherein each R^(A) is independently hydrogen or C₁₋₆ alkyl.R³ is Selected from One of the Following Groups (2a)-(2Aa):

-   -   (2a) R³ is one of groups (R³-a) through (R³-g) as defined above.    -   (2b) R³ is one of groups (R³-b) through (R³-g) as defined above.

-   -   (2u) Any one of groups (2l)-(2t), wherein R³⁵ is hydrogen or        methyl.    -   (2v) Any one of groups (2l)-(2t), wherein R³⁵ is methyl.    -   (2w) Any one of groups (2l)-(2t), wherein R³⁵ is hydrogen.    -   (2x) R³ is

-   -   (2y) Group (2x) wherein R³³ is C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆        haloalkyl, C₃₋₈ cycloalkyl, aryl, or arylC₁₋₆ alkyl.    -   (2z) Group (2x) wherein R³³ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₈        cycloalkyl, phenyl, or benzyl.    -   (2aa) Group (2x) wherein R³³ is C₁₋₄ alkyl, C₁₋₄ haloalkyl, C₅₋₆        cycloalkyl, phenyl, or benzyl.        Q is Selected from One of the Following Groups (3a)-(3e):    -   (3a) Q is —O— or —N(R^(Q))—.    -   (3b) Q is —O— or —N(H)—.    -   (3c) Q is —O—.    -   (3d) Q is —N(R^(Q))—.    -   (3e) Q is —N(H)—.        n and R³² are Selected from One of the Following Groups        (4a)-(4x):    -   (4a) n is 0.    -   (4b) n is 0 or 1 and R³² is as defined for formula (I).    -   (4c) n is 0 or 1 and R³² is halogen, cyano, nitro, C₁₋₆ alkyl,        or C₁₋₆ haloalkyl.    -   (4d) n is 0 or 1 and R³² is halogen, C₁₋₆ alkyl, or C₁₋₆        haloalkyl.    -   (4e) n is 0 or 1 and each R³² is —OR³⁴, —SR³⁴, —N(R³⁴)₂,        —C(O)R³⁴, —C(O)OR³⁴, —C(O)N(R³⁴)₂, —S(O)₂R³⁴, —OC(O)R³⁴,        —OC(O)OR³⁴, —OC(O)N(R³⁴)₂, —N(R³⁴)C(O)R³⁴, —N(R³⁴)C(O)OR³⁴, or        —N(R³⁴)C(O)N(R³⁴)₂, wherein each R³⁴ is independently hydrogen        or C₁₋₆ alkyl.    -   (4f) n is 0 or 1 and R³² is —OR³⁴, —SR³⁴, —N(R³⁴)₂, wherein each        R³⁴ is independently hydrogen or C₁₋₆ alkyl.    -   (4g) n is 0 or 1 and R³² is —C(O)R³⁴, —C(O)OR³⁴, —C(O)N(R³⁴)₂,        or —S(O)₂R³⁴, wherein each R³⁴ is independently hydrogen or C₁₋₆        alkyl.    -   (4h) n is as defined for formula (I) and each R³² is        independently halogen, cyano, nitro, C₁₋₆ alkyl, or C₁₋₆        haloalkyl.    -   (4i) n is as defined for formula (I) and each R³² is        independently halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (4j) n is as defined for formula (I) and each R³² is        independently —OR³⁴, —SR³⁴, —N(R³⁴)₂, —C(O)R³⁴, —C(O)OR³⁴,        —C(O)N(R³⁴)₂, —S(O)₂R³⁴, —OC(O)R³⁴, —OC(O)OR³⁴, —OC(O)N(R³⁴)₂,        —N(R³⁴)C(O)R³⁴, —N(R³⁴)C(O)OR³⁴, or —N(R³⁴)C(O)N(R³⁴)₂, wherein        each R³⁴ is independently hydrogen or C₁₋₆ alkyl.    -   (4k) n is as defined for formula (I) and each R³² is        independently —OR³⁴, —SR³⁴, —N(R³⁴)₂, wherein each R³⁴ is        independently hydrogen or C₁₋₆ alkyl.    -   (4l) n is as defined for formula (I) and each R³² is        independently —C(O)R³⁴, —C(O)OR³⁴, —C(O)N(R³⁴)₂, or —S(O)₂R³⁴,        wherein each R³⁴ is independently hydrogen or C₁₋₆ alkyl.    -   (4m) n is 1 or 2 and each R³² is as defined for formula (I).    -   (4n) n is 1 or 2 and each R³² is independently halogen, cyano,        nitro, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (4o) n is 1 or 2 and each R³² is independently halogen, C₁₋₆        alkyl, or C₁₋₆ haloalkyl.    -   (4p) n is 1 or 2 and each R³² is independently —OR³⁴, —SR³⁴,        —N(R³⁴)₂, —C(O)R³⁴, —C(O)OR³⁴, —C(O)N(R³⁴)₂, —S(O)₂R³⁴,        —OC(O)R³⁴, —OC(O)OR³⁴, —OC(O)N(R³⁴)₂, —N(R³⁴)C(O)R³⁴,        —N(R³⁴)C(O)OR³⁴, or —N(R³⁴)C(O)N(R³⁴)₂, wherein each R³⁴ is        independently hydrogen or C₁₋₆ alkyl.    -   (4q) n is 1 or 2 and each R³² is independently —OR³⁴, —SR³⁴,        —N(R³⁴)₂, wherein each R³⁴ is independently hydrogen or C₁₋₆        alkyl.    -   (4r) n is 1 or 2 and each R³² is independently —C(O)R³⁴,        —C(O)OR³⁴, —C(O)N(R³⁴)₂, or —S(O)₂R³⁴, wherein each R³⁴ is        independently hydrogen or C₁₋₆ alkyl.    -   (4s) n is 1 and R³² is as defined for formula (I).    -   (4t) n is 1 and R³² is halogen, cyano, nitro, C₁₋₆ alkyl, or        C₁₋₆ haloalkyl.    -   (4u) n is 1 and R³² is halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (4v) n is 1 and R³² is —OR³⁴, —SR³⁴, —N(R³⁴)₂, —C(O)R³⁴,        —C(O)OR³⁴, —C(O)N(R³⁴)₂, —S(O)₂R³⁴, —OC(O)R³⁴, —OC(O)OR³⁴,        —OC(O)N(R³⁴)₂, —N(R³⁴)C(O)R³⁴, —N(R³⁴)C(O)OR³⁴, or        —N(R³⁴)C(O)N(R³⁴)₂, wherein each R³⁴ is independently hydrogen        or C₁₋₆ alkyl.    -   (4w) n is 1 and R³² is-OR³⁴, —SR³⁴, —N(R³⁴)₂, wherein each R³⁴        is independently hydrogen or C₁₋₆ alkyl.    -   (4x) n is 1 and R³² is —C(O)R³⁴, —C(O)OR³⁴, —C(O)N(R³⁴)₂, or        —S(O)₂R³⁴, wherein each R³⁴ is independently hydrogen or C₁₋₆        alkyl.        R³³ is Selected from One of the Following Groups (5a)-(5t):    -   (5a) R³³ is C₁₋₆ alkyl, C₂₋₆ alkenyl, arylC₁₋₆ alkyl, or        heteroarylC₁₋₆ alkyl, wherein the arylalkyl and heteroarylalkyl        are optionally substituted with one, two, three, or four groups        that are each independently halogen, cyano, nitro, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰,        —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —O C(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂,        —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or —N(R²⁰)C(O)N(R²⁰)₂, wherein        each R²⁰ is independently hydrogen or C₁₋₆ alkyl.    -   (5b) R³³ is C₁₋₆ alkyl, C₂₋₆ alkenyl, arylC₁₋₆ alkyl, or        heteroarylC₁₋₆ alkyl, wherein the arylalkyl and heteroarylalkyl        are each substituted with one, two, three, or four groups that        are each independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆        haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰,        —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —O C(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂,        —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰ or —N(R²⁰)C(O)N(R²⁰)₂, wherein        each R²⁰ is independently hydrogen or C₁₋₆ alkyl.    -   (5c) R³³ is C₁₋₆ alkyl, C₂₋₆ alkenyl, arylC₁₋₆ alkyl, or        heteroarylC₁₋₆ alkyl, wherein the arylalkyl and heteroarylalkyl        are optionally substituted with one or two groups that are each        independently halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (5d) R³³ is C₁₋₆ alkyl, C₂₋₆ alkenyl, arylC₁₋₆ alkyl, or        heteroarylC₁₋₆ alkyl, wherein the arylalkyl and heteroarylalkyl        are each substituted with one or two groups that are each        independently halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (5e) R³³ is C₂₋₆ alkenyl, arylC₁₋₆ alkyl, or heteroarylC₁₋₆        alkyl, wherein the arylalkyl and heteroarylalkyl are optionally        substituted with one, two, three, or four groups that are each        independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,        —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, —C(O)N(R²⁰)₂,        —S(O)₂R²⁰, —O C(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂,        —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or —N(R²⁰)C(O)N(R²⁰)₂, wherein        each R²⁰ is independently hydrogen or C₁₋₆ alkyl.    -   (5f) R³³ is C₂₋₆ alkenyl, arylC₁₋₆ alkyl, or heteroarylC₁₋₆        alkyl, wherein the arylalkyl and heteroarylalkyl are each        substituted with one, two, three, or four groups that are each        independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,        —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, —C(O)N(R²⁰)₂,        —S(O)₂R²⁰, —O C(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂,        —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or —N(R²⁰)C(O)N(R²⁰)₂, wherein        each R²⁰ is independently hydrogen or C₁₋₆ alkyl.    -   (5g) R³³ is C₂₋₆ alkenyl, arylC₁₋₆ alkyl, or heteroarylC₁₋₆        alkyl, wherein the arylalkyl and heteroarylalkyl are optionally        substituted with one or two groups that are each independently        halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (5h) R³³ is C₂₋₆ alkenyl, arylC₁₋₆ alkyl, or heteroarylC₁₋₆        alkyl, wherein the arylalkyl and heteroarylalkyl are each        substituted with one or two groups that are each independently        halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (5i) R³³ is arylC₁₋₆ alkyl or heteroarylC₁₋₆ alkyl, each are        optionally substituted with one, two, three, or four groups that        are each independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆        haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰,        —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —OC(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂,        —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or —N(R²⁰)C(O)N(R²⁰)₂, wherein        each R²⁰ is independently hydrogen or C₁₋₆ alkyl.    -   (5j) R³³ is arylC₁₋₆ alkyl or heteroarylC₁₋₆ alkyl, each are        each substituted with one, two, three, or four groups that are        each independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆        haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰,        —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —O C(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂,        —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or —N(R²⁰)C(O)N(R²⁰)₂, wherein        each R²⁰ is independently hydrogen or C₁₋₆ alkyl    -   (5k) R³³ is arylC₁₋₆ alkyl or heteroarylC₁₋₆ alkyl, each        optionally substituted with one or two groups that are each        independently halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (5l) R³³ is arylC₁₋₆ alkyl or heteroarylC₁₋₆ alkyl, each        substituted with one or two groups that are each independently        halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (5m) R³³ is arylC₁₋₆ alkyl optionally substituted with one, two,        three, or four groups that are each independently halogen,        cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰,        —N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —O        C(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰,        —N(R²⁰)C(O)OR²⁰, or —N(R²⁰)C(O)N(R²⁰)₂, wherein each R²⁰ is        independently hydrogen or C₁₋₆ alkyl.    -   (5n) R³³ is arylC₁₋₆ alkyl substituted with one, two, three, or        four groups that are each independently halogen, cyano, nitro,        C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰,        —C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —OC(O)R²⁰, —OC(O)OR²⁰,        —OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or        —N(R²⁰)C(O)N(R²⁰)₂, wherein each R²⁰ is independently hydrogen        or C₁₋₆ alkyl    -   (5o) R³³ is arylC₁₋₆ alkyl optionally substituted with one or        two groups that are each independently halogen, C₁₋₆ alkyl, or        C₁₋₆ haloalkyl.    -   (5p) R³³ is arylC₁₋₆ alkyl substituted with one or two groups        that are each independently halogen, C₁₋₆ alkyl, or C₁₋₆        haloalkyl.    -   (5q) R³³ is benzyl optionally substituted with one, two, three,        or four groups that are each independently halogen, cyano,        nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂,        —C(O)R²⁰, —C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —OC(O)R²⁰,        —OC(O)OR²⁰, —OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or        —N(R²⁰)C(O)N(R²⁰)₂, wherein each R²⁰ is independently hydrogen        or C₁₋₆ alkyl.    -   (5r) R³³ is benzyl substituted with one, two, three, or four        groups that are each independently halogen, cyano, nitro, C₁₋₆        alkyl, C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰,        —C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —OC(O)R²⁰, —OC(O)OR²⁰,        —OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or        —N(R²⁰)C(O)N(R²⁰)₂, wherein each R²⁰ is independently hydrogen        or C₁₋₆ alkyl.    -   (5s) R³³ is benzyl optionally substituted with one or two groups        that are each independently halogen, C₁₋₆ alkyl, or C₁₋₆        haloalkyl.    -   (5t) R³³ is benzyl substituted with one or two groups that are        each independently halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.

Particular embodiments of this aspect of the invention include compoundsof any one of the formulae (I), (Ia), and (Ib), each as defined in eachof the following rows, wherein each entry is a group number as definedabove (e.g., (3c) refers to Q is —O—), a dash “-” indicates that thevariable is as defined for formula (I) or defined according to any oneof the applicable variable definitions (1a)-(5t) [e.g., when R¹ is adash, it can be either as defined for Formula (I) or any one ofdefinitions (1a)-(1ii)]; and an “x” indicates that the variable is notapplicable to the particular embodiment (e.g., when R³ is (2o), then Qand R³³ are not necessary):

n & R¹ R³ R³³ R³² Q (1)-1 1h 2b 5a 4c 3a (1)-2 1n 2b 5a 4c 3a (1)-3 1p2b 5a 4c 3a (1)-4 1aa 2b 5a 4c 3a (1)-5 1h 2b 5e 4c 3a (1)-6 1n 2b 5e 4c3a (1)-7 1p 2b 5e 4c 3a (1)-8 1aa 2b 5e 4c 3a (1)-9 1h 2b 5i 4c 3a(1)-10 1n 2b 5i 4c 3a (1)-11 1p 2b 5i 4c 3a (1)-12 1aa 2b 5i 4c 3a(1)-13 1h 2b 5q 4c 3a (1)-14 1n 2b 5q 4c 3a (1)-15 1p 2b 5q 4c 3a (1)-161aa 2b 5q 4c 3a (1)-17 1h 2c 5a 4c 3a (1)-18 1n 2c 5a 4c 3a (1)-19 1p 2c5a 4c 3a (1)-20 1aa 2c 5a 4c 3a (1)-21 1h 2c 5e 4c 3a (1)-22 1n 2c 5e 4c3a (1)-23 1p 2c 5e 4c 3a (1)-24 1aa 2c 5e 4c 3a (1)-25 1h 2c 5i 4c 3a(1)-26 1n 2c 5i 4c 3a (1)-27 1p 2c 5i 4c 3a (1)-28 1aa 2c 5i 4c 3a(1)-29 1h 2c 5q 4c 3a (1)-30 1n 2c 5q 4c 3a (1)-31 1p 2c 5q 4c 3a (1)-321aa 2c 5q 4c 3a (1)-33 1h 2b 5a 4n 3a (1)-34 1n 2b 5a 4n 3a (1)-35 1p 2b5a 4n 3a (1)-36 1aa 2b 5a 4n 3a (1)-37 1h 2b 5e 4n 3a (1)-38 1n 2b 5e 4n3a (1)-39 1p 2b 5e 4n 3a (1)-40 1aa 2b 5e 4n 3a (1)-41 1h 2b 5i 4n 3a(1)-42 1n 2b 5i 4n 3a (1)-43 1p 2b 5i 4n 3a (1)-44 1aa 2b 5i 4n 3a(1)-45 1h 2b 5q 4n 3a (1)-46 1n 2b 5q 4n 3a (1)-47 1p 2b 5q 4n 3a (1)-481aa 2b 5q 4n 3a (1)-49 1h 2c 5a 4n 3a (1)-50 1n 2c 5a 4n 3a (1)-51 1p 2c5a 4n 3a (1)-52 1aa 2c 5a 4n 3a (1)-53 1h 2c 5e 4n 3a (1)-54 1n 2c 5e 4n3a (1)-55 1p 2c 5e 4n 3a (1)-56 1aa 2c 5e 4n 3a (1)-57 1h 2c 5i 4n 3a(1)-58 1n 2c 5i 4n 3a (1)-59 1p 2c 5i 4n 3a (1)-60 1aa 2c 5i 4n 3a(1)-61 1h 2c 5q 4n 3a (1)-62 1n 2c 5q 4n 3a (1)-63 1p 2c 5q 4n 3a (1)-641aa 2c 5q 4n 3a (1)-65 1h 2b 5a 4c 3c (1)-66 1n 2b 5a 4c 3c (1)-67 1p 2b5a 4c 3c (1)-68 1aa 2b 5a 4c 3c (1)-69 1h 2b 5e 4c 3c (1)-70 1n 2b 5e 4c3c (1)-71 1p 2b 5e 4c 3c (1)-72 1aa 2b 5e 4c 3c (1)-73 1h 2b 5i 4c 3c(1)-74 1n 2b 5i 4c 3c (1)-75 1p 2b 5i 4c 3c (1)-76 1aa 2b 5i 4c 3c(1)-77 1h 2b 5q 4c 3c (1)-78 1n 2b 5q 4c 3c (1)-79 1p 2b 5q 4c 3c (1)-801aa 2b 5q 4c 3c (1)-81 1h 2c 5a 4c 3c (1)-82 1n 2c 5a 4c 3c (1)-83 1p 2c5a 4c 3c (1)-84 1aa 2c 5a 4c 3c (1)-85 1h 2c 5e 4c 3c (1)-86 1n 2c 5e 4c3c (1)-87 1p 2c 5e 4c 3c (1)-88 1aa 2c 5e 4c 3c (1)-89 1h 2c 5i 4c 3c(1)-90 1n 2c 5i 4c 3c (1)-91 1p 2c 5i 4c 3c (1)-92 1aa 2c 5i 4c 3c(1)-93 1h 2c 5q 4c 3c (1)-94 1n 2c 5q 4c 3c (1)-95 1p 2c 5q 4c 3c (1)-961aa 2c 5q 4c 3c (1)-97 1h 2b 5a 4n 3c (1)-98 1n 2b 5a 4n 3c (1)-99 1p 2b5a 4n 3c (1)-100 1aa 2b 5a 4n 3c (1)-101 1h 2b 5e 4n 3c (1)-102 1n 2b 5e4n 3c (1)-103 1p 2b 5e 4n 3c (1)-104 1aa 2b 5e 4n 3c (1)-105 1h 2b 5i 4n3c (1)-106 1n 2b 5i 4n 3c (1)-107 1p 2b 5i 4n 3c (1)-108 1aa 2b 5i 4n 3c(1)-109 1h 2b 5q 4n 3c (1)-110 1n 2b 5q 4n 3c (1)-111 1p 2b 5q 4n 3c(1)-112 1aa 2b 5q 4n 3c (1)-113 1h 2c 5a 4n 3c (1)-114 1n 2c 5a 4n 3c(1)-115 1p 2c 5a 4n 3c (1)-116 1aa 2c 5a 4n 3c (1)-117 1h 2c 5e 4n 3c(1)-118 1n 2c 5e 4n 3c (1)-119 1p 2c 5e 4n 3c (1)-120 1aa 2c 5e 4n 3c(1)-121 1h 2c 5i 4n 3c (1)-122 1n 2c 5i 4n 3c (1)-123 1p 2c 5i 4n 3c(1)-124 1aa 2c 5i 4n 3c (1)-125 1h 2c 5q 4n 3c (1)-126 1n 2c 5q 4n 3c(1)-127 1p 2c 5q 4n 3c (1)-128 1aa 2c 5q 4n 3c (1)-129 1h 2b — — —(1)-130 1n 2b — — — (1)-131 1p 2b — — — (1)-132 1aa 2b — — — (1)-133 1h2c — — — (1)-134 1n 2c — — — (1)-135 1p 2c — — — (1)-136 1aa 2c — — —(1)-137 1h 2b — — 3c (1)-138 1n 2b — — 3c (1)-139 1p 2b — — 3c (1)-1401aa 2b — — 3c (1)-141 1h 2c — — 3c (1)-142 1n 2c — — 3c (1)-143 1p 2c —— 3c (1)-144 1aa 2c — — 3c (1)-145 1h 2b — — 3a (1)-146 1n 2b — — 3a(1)-147 1p 2b — — 3a (1)-148 1aa 2b — — 3a (1)-149 1h 2c — — 3a (1)-1501n 2c — — 3a (1)-151 1p 2c — — 3a (1)-152 1aa 2c — — 3a (1)-153 — — 5a —3a (1)-154 — — 5e — 3a (1)-155 — — 5i — 3a (1)-156 — — 5q — 3a (1)-157 —— 5a — 3c (1)-158 — — 5e — 3a (1)-159 — — 5i — 3a (1)-160 — — 5q — 3a(1)-161 — 2b 5a — 3a (1)-162 — 2b 5e — 3a (1)-163 — 2b 5i — 3a (1)-164 —2b 5q — 3a (1)-165 — 2b 5a — 3c (1)-166 — 2b 5e — 3a (1)-167 — 2b 5i —3a (1)-168 — 2b 5q — 3a (1)-169 — 2c 5a — 3a (1)-170 — 2c 5e — 3a(1)-171 — 2c 5i — 3a (1)-172 — 2c 5q — 3a (1)-173 — 2c 5a — 3c (1)-174 —2c 5e — 3a (1)-175 — 2c 5i — 3a (1)-176 — 2c 5q — 3a (1)-177 1h 2o x 4cx (1)-178 1n 2o x 4c x (1)-179 1p 2o x 4c x (1)-180 1aa 2o x 4c x(1)-181 1h 2r x 4c x (1)-182 1n 2r x 4c x (1)-183 1p 2r x 4c x (1)-1841aa 2r x 4c x (1)-185 1h 2o x 4n x (1)-186 1n 2o x 4n x (1)-187 1p 2o x4n x (1)-188 1aa 2o x 4n x (1)-189 1h 2r x 4n x (1)-190 1n 2r x 4n x(1)-191 1p 2r x 4n x (1)-192 1aa 2r x 4n x (1)-193 1h 2o x — x (1)-1941n 2o x — x (1)-195 1p 2o x — x (1)-196 1aa 2o x — x (1)-197 1h 2r x — x(1)-198 1n 2r x — x (1)-199 1p 2r x — x (1)-200 1aa 2r x — x

In one embodiment, the compound is one of the compounds in Table A:

TABLE A

1-(6-ethoxynaphthalen-2-yl)-3-isopropylimidazo[1,5- a]pyrazin-8-amine;

3-(6-isopropoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

1-isopropyl-3-(6-propoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

1-isopropyl-3-(6-methoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-ethoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(1H-indol-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin- 4-amine;

1-isopropyl-3-(4-methyl-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(4-chloro-1H-indol-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

1-isopropyl-3-(5-methoxy-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

3-(6-methoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-ethoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-ethoxynaphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

3-(6-isopropoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

1-(piperidin-4-ylmethyl)-3-(6-propoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-(benzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-butoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-(allyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-(2-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine

3-(6-(3-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-(4-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-(benzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-(allyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-butoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-isobutoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-isobutoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-(2-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-(3-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-(2,5-dimethylbenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

1-isopropyl-3-(6-(2-methylbenzyloxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

1-isopropyl-3-(6-(2-methyl-5-(trifluoromethyl)benzyloxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-(3-chloro-4-(2,2,2-trifluoroethyl)benzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-(3-chloro-5-fluorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

1-isopropyl-3-(6-(1-phenylethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-(4-tert-butylbenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

1-isopropyl-3-(6-(pyridin-4-ylmethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

3-(6-(4-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;

6-(4-amino-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N,N-dimethylquinolin-2-amine;

3-tert-butyl-1-(6-ethoxynaphthalen-2-yl)imidazo[1,5- a]pyrazin-8-amine;

3-tert-butyl-1-(6-methoxynaphthalen-2-yl)imidazo[1,5- a]pyrazin-8-amine;and a pharmaceutically acceptable salts thereof.

In another embodiment, the present disclosure provides compounds of theformula (III),

or a pharmaceutically acceptable salt thereof, wherein

-   X, Y, and Z are defined by either: (i) X is N, Y is C, and Z is N;    or (ii) X is C, Y is N, and Z is C(H);-   R¹ is C₂₋₆ alkyl or —C₁₋₄ alkyl-R¹², wherein    -   R¹² is a monocyclic heterocyclyl optionally substituted by one,        two, or three groups that are each independently halogen, cyano,        nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR, —SR, —NR₂, —C(O)R,        —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂,        —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂;-   R³ is one of the formulas,

wherein

-   -   n is 0, 1, or 2;    -   Q is —O— or —N(R^(Q))—, wherein R^(Q) is hydrogen or C₁₋₆ alkyl;        and    -   R³³ is C₁₋₆ alkyl, C₂₋₆ alkenyl, arylC₁₋₆ alkyl, or        heteroarylC₁₋₆ alkyl wherein the arylalkyl and heteroarylalkyl        are optionally substituted with one, two, three, or four groups        that are each independently halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl,        —OR²⁰, —SR²⁰, or —N(R²⁰)₂, wherein each R²⁰ is independently        hydrogen or C₁₋₆ alkyl.    -   each R³² is independently halogen, C₁₋₆ alkyl, or —OR³⁴, wherein        R³⁴ is C₁₋₆ alkyl;    -   and    -   R³⁵ is hydrogen or C₁₋₆ alkyl;

-   and

-   each R is independently hydrogen or C₁₋₆ alkyl.

The disclosure further comprises subgenera of formula (III) in which thesubstituents are selected as any and all combinations of one or more ofstructural formula (III), n, Q, R¹, R³, R³², and R³³ as defined herein,including without limitation, the following:

Structural Formula III is One of Formulae (IIIa)-(IIIb):

R¹ is selected from one of the following groups (8a)-(8u):

-   (8a) R¹ is C₂₋₆ alkyl.-   (8b) R¹ is C₂₋₄ alkyl.-   (8c) R¹ is isopropyl or t-butyl.-   (8d) R¹ is t-butyl.-   (8e) R¹ is isopropyl.-   (8f) R¹ is —C₁₋₄ alkyl-R¹².-   (8g) R¹ is —C₁₋₂ alkyl-R¹².-   (8h) R¹ is —CH₂—R¹²-   (8i) Any one of groups (8f)-(8h), wherein R¹² is monocyclic    heterocyclyl optionally substituted by one or two groups that are    each independently halogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂,    —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or    —N(R)C(O)NR₂.-   (8j) Any one of groups (8f)-(8h), R¹² is piperidinyl or    tetrahydropyranyl, each optionally substituted by one or two groups    that are each independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆    haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,    —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or    —N(R)C(O)NR₂.-   (8k) Any one of groups (8f)-(8h), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,    —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R,    —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂.-   (8l) Any one of groups (8f)-(8h), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A), —C(O)N(R^(A))₂,    —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,    —N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,    wherein each R^(A) is independently hydrogen, C₁₋₆ alkyl, C₂₋₆    alkenyl, C₁₋₆ haloalkyl, C₃₋₈ cycloalkyl, heterocyclyl, aryl,    arylC₁₋₆ alkyl, heteroaryl, or heteroarylC₁₋₆ alkyl.-   (8m) Any one of groups (8f)-(8h), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,    —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or    —N(R)C(O)NR₂.-   (8n) Any one of groups (8f)-(8h), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A), —C(O)N(R^(A))₂,    —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,    —N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,    wherein each R^(A) is independently hydrogen or C₁₋₆ alkyl.-   (8o) Any one of groups (8f)-(8h), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, or —S(O)₂R.-   (8p) Any one of groups (8f)-(8h), wherein R¹² is piperidinyl    optionally substituted by one or two groups that are each    independently C₁₋₆ alkyl, —C(O)R^(A), or —S(O)₂R^(A), wherein each    R^(A) is independently hydrogen or C₁₋₆ alkyl.-   (8q) Any one of groups (8f)-(8h), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂,—S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or—N(R)C(O)NR₂.

-   (8r) Any one of groups (8f)-(8h), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A),—C(O)N(R^(A))₂, —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,—N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,wherein each R^(A) is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₁₋₆ haloalkyl, C₃₋₈ cycloalkyl, heterocyclyl, aryl, arylC₁₋₆ alkyl,heteroaryl, or heteroarylC₁₋₆ alkyl.

-   (8s) Any one of groups (8f)-(8h), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A),—C(O)N(R^(A))₂, —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,—N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,wherein each R^(A) is independently hydrogen or C₁₋₆ alkyl.

-   (8t) Any one of groups (8f)-(8h), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, or—S(O)₂R.

-   (8u) Any one of groups (8f)-(8h), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), or —S(O)₂R^(A),wherein each R^(A) is independently hydrogen or C₁₋₆ alkyl.R³ is Selected from One of the Following Groups (9a)-(9g):

-   -   (9e) Any one of groups (9b)-(9d), wherein R³⁵ is hydrogen or        methyl.    -   (9f) Any one of groups (9b)-(9d), wherein R³⁵ is methyl.    -   (9g) Any one of groups (9b)-(9d), wherein R³⁵ is hydrogen.        Q is Selected from One of the Following Groups (10a)-(10d):    -   (10a) Q is —O— or —N(H)—.    -   (10b) Q is —O—.    -   (10c) Q is —N(R^(Q))—.    -   (10d) Q is —N(H)—.        n and R³² are Selected from One of the Following Groups        (11a)-(11c):    -   (11a) n is 0.    -   (11b) n is 0 or 1 and R³² is as defined for formula (III).    -   (11c) n is 1 or 2 and R³² is as defined for formula (III).        R³³ is Selected from One of the Following Groups (12a)-(12i):    -   (12a) R³³ is C₁₋₆ alkyl, C₂₋₆ alkenyl, arylC₁₋₆ alkyl, or        heteroarylC₁₋₆ alkyl, wherein the arylalkyl and heteroarylalkyl        are optionally substituted with one or two groups that are each        independently halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (12b) R³³ is C₁₋₆ alkyl, C₂₋₆ alkenyl, arylC₁₋₆ alkyl, or        heteroarylC₁₋₆ alkyl, wherein the arylalkyl and heteroarylalkyl        are each substituted with one or two groups that are each        independently halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (12c) R³³ is C₂₋₆ alkenyl, arylC₁₋₆ alkyl, or heteroarylC₁₋₆        alkyl, wherein the arylalkyl and heteroarylalkyl are optionally        substituted with one or two groups that are each independently        halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (12d) R³³ is C₂₋₆ alkenyl, arylC₁₋₆ alkyl, or heteroarylC₁₋₆        alkyl, wherein the arylalkyl and heteroarylalkyl are each        substituted with one or two groups that are each independently        halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (12e) R³³ is arylC₁₋₆ alkyl or heteroarylC₁₋₆ alkyl, each        optionally substituted with one or two groups that are each        independently halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (12f) R³³ is arylC₁₋₆ alkyl or heteroarylC₁₋₆ alkyl, each        substituted with one or two groups that are each independently        halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.    -   (12g) R³³ is arylC₁₋₆ alkyl optionally substituted with one or        two groups that are each independently halogen, C₁₋₆ alkyl, or        C₁₋₆ haloalkyl.    -   (12h) R³³ is arylC₁₋₆ alkyl substituted with one or two groups        that are each independently halogen, C₁₋₆ alkyl, or C₁₋₆        haloalkyl.    -   (12i) R³³ is benzyl optionally substituted with one or two        groups that are each independently halogen, C₁₋₆ alkyl, or C₁₋₆        haloalkyl.    -   (12j) R³³ is benzyl substituted with one or two groups that are        each independently halogen, C₁₋₆ alkyl, or C₁₋₆ haloalkyl.

Particular embodiments of this aspect of the invention include compoundsof any one of the formulae (III), (IIIa), and (IIIb), each as defined ineach of the following rows, wherein each entry is a group number asdefined above (e.g., (10b) refers to Q is —O—), and a dash “-” indicatesthat the variable is as defined for formula (III) or defined accordingto any one of the applicable variable definitions (8a)-(12j) [e.g., whenR¹ is a dash, it can be either as defined for Formula (III) or any oneof definitions (8a)-(8u)]; and an “x” indicates that the variable is notapplicable to the particular embodiment (e.g., when R³ is (9c), then Qand R³³ are not necessary):

n & R¹ R³ R³³ R³² Q (3)-1 8a 9a 12a — 10b (3)-2 8f 9a 12a — 10b (3)-3 8g9a 12a — 10b (3)-4 8h 9a 12a — 10b (3)-5 8m 9a 12a — 10b (3)-6 8a 9a 12c— 10b (3)-7 8f 9a 12c — 10b (3)-8 8g 9a 12c — 10b (3)-9 8h 9a 12c — 10b(3)-10 8m 9a 12c — 10b (3)-11 8a 9a 12e — 10b (3)-12 8f 9a 12e — 10b(3)-13 8g 9a 12e — 10b (3)-14 8h 9a 12e — 10b (3)-15 8m 9a 12e — 10b(3)-16 8a 9a 12i — 10b (3)-17 8f 9a 12i — 10b (3)-18 8g 9a 12i — 10b(3)-19 8h 9a 12i — 10b (3)-20 8m 9a 12i — 10b (3)-21 8a 9c x — x (3)-228f 9c x — x (3)-23 8g 9c x — x (3)-24 8h 9c x — x (3)-25 8m 9c x — x(3)-26 8a 9d x — x (3)-27 8f 9d x — x (3)-28 8g 9d x — x (3)-29 8h 9d x— x (3)-30 8m 9d x — x (3)-31 8a 9a — — — (3)-32 8f 9a — — — (3)-33 8g9a — — — (3)-34 8h 9a — — — (3)-35 8m 9a — — — (3)-36 8a 9a — — 10b(3)-37 8f 9a — — 10b (3)-38 8g 9a — — 10b (3)-39 8h 9a — — 10b (3)-40 8m9a — — 10b (3)-41 — — 12a — 10b (3)-42 — — 12c — 10b (3)-43 — — 12e —10b (3)-44 — — 12i — 10b (3)-45 — 9a 12a — 10b (3)-46 — 9a 12c — 10b(3)-47 — 9a 12e — 10b (3)-48 — 9a 12i — 10b

Particular embodiments of this aspect of the invention include compoundsof any one of the formula (IIIb), each as defined in each of thefollowing rows, wherein each entry is a group number as defined above(e.g., (10b) refers to Q is —O—). In addition, any one of groups(8f)-(8h) can be either as defined for Formula (IIIb) or any one ofdefinitions (8f)-(8u):

n & R¹ R³ R³³ R³² Q (3)-49 8f 9a 12a 11a 10a (3)-50 8g 9a 12a 11a 10a(3)-51 8h 9a 12a 11a 10a (3)-52 8f 9a 12b 11a 10a (3)-53 8g 9a 12b 11a10a (3)-54 8h 9a 12b 11a 10a (3)-55 8f 9a 12a 11a 10b (3)-56 8g 9a 12a11a 10b (3)-57 8h 9a 12a 11a 10b (3)-58 8f 9a 12b 11a 10b (3)-59 8g 9a12b 11a 10b (3)-60 8h 9a 12b 11a 10b (3)-61 8f 9a 12a 11b 10a (3)-62 8g9a 12a 11b 10a (3)-63 8h 9a 12a 11b 10a (3)-64 8f 9a 12b 11b 10a (3)-658g 9a 12b 11b 10a (3)-66 8h 9a 12b 11b 10a (3)-67 8f 9a 12a 11b 10b(3)-68 8g 9a 12a 11b 10b (3)-69 8h 9a 12a 11b 10b (3)-70 8f 9a 12b 11b10b (3)-71 8g 9a 12b 11b 10b (3)-72 8h 9a 12b 11b 10b

In an embodiment of the preceding embodiments of formulas (I) and (III),the compound is not

Methods for Use

Clinical toxoplasmosis is caused by the actively dividing tachyzoiteform of the parasite, which exits its host cell and invades a new cellevery few days. TgCDPK1 has a unique ATP-binding site with a smallgatekeeper residue, as opposed to the large gatekeeper residues presentin mammalian protein kinases. This key difference in gatekeeper residuesbetween mammalian kinases and TgCDPK1 allowed us to use bumped kinaseinhibitors (BKIs) to selectively inhibit TgCDPK1 without untowardeffects on the mammalian host cell.

Accordingly, the disclosure provides methods for treating anapicomplexan protozoan related disease comprising providing to a patientin need of such treatment a therapeutically effective amount of either(i) a bumped kinase inhibitor or (ii) a pharmaceutical compositioncomprising a bumped kinase inhibitor and a pharmaceutically acceptableexcipient, carrier, or diluent.

In one embodiment, the bumped kinase inhibitor is selected from one ofthe compounds of FIG. 14, or a pharmaceutically acceptable salt thereof.

In another embodiment, the bumped kinase inhibitor is according to ofthe formula (I) as defined above, or any embodiment thereof.

In another embodiment, the bumped kinase inhibitor is of the formula(II),

or a pharmaceutically acceptable salt thereof, wherein

-   X, Y, and Z are defined by either: (i) X is N, Y is C, and Z is N;    or (ii) X is C, Y is N, and Z is C(H);-   R¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, —C₁₋₆    alkyl-R¹², C₃₋₈ cycloalkyl, monocyclic heterocyclyl, monocyclic    heteroaryl, or phenyl, wherein    -   the cycloalkyl, monocyclic heterocyclyl, monocyclic heteroaryl,        and phenyl groups are each optionally substituted with one or        two R¹¹ groups;    -   each R¹¹ is independently C₁₋₆ alkyl, C₁₋₆ haloalkyl, —C(O)R,        —C(O)OR, —C(O)NR₂, —S(O)₂NR₂, or —S(O)₂R;    -   and    -   R¹² is —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,        —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR,        —N(R)C(O)NR₂, phenyl, monocyclic heteroaryl, C₃₋₈ cycloalkyl, or        monocyclic heterocyclyl, wherein the phenyl, heteroaryl, C₃₋₈        cycloalkyl, and heterocyclyl groups are each optionally        substituted by one, two, or three groups that are each        independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,        —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R,        —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂;-   L is a bond or —CH₂—;-   R³ is aryl or heteroaryl, each optionally substituted with one, two,    or three R³¹ groups,-   wherein    -   each R³¹ is independently halogen, cyano, nitro, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, aryl, heteroaryl, C₃₋₈ cycloalkyl, heterocyclyl,        arylC₁₋₆ alkyl, heteroarylC₁₋₆ alkyl, —OR, —SR, —NR₂, —C(O)R,        —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂,        —N(R)C(O)R, —N(R)C(O)OR, —N(R)C(O)NR₂ or —N(R)S(O)₂R, wherein        the aryl, heteroaryl, C₃₋₈ cycloalkyl, heterocyclyl, arylalkyl,        and heteroarylalkyl groups are optionally substituted with one        or two R¹⁰ groups;    -   each R is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆        haloalkyl, C₃₋₈ cycloalkyl, heterocyclyl, aryl, arylC₁₋₆ alkyl,        heteroaryl, or heteroarylC₁₋₆ alkyl wherein the alkyl, aryl,        arylalkyl, heteroaryl, and heteroarylalkyl are optionally        substituted with one, two, three, or four R¹⁰ groups;    -   and    -   each R¹⁰ is independently halogen, cyano, nitro, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, —OR⁰, —SR⁰, —N(R⁰)₂, —C(O)R⁰, —C(O)OR⁰,        —C(O)N(R⁰)₂, —S(O)₂R, —OC(O)R⁰, —OC(O)OR⁰, —OC(O)N(R⁰)₂,        —N(R⁰)C(O)R, —N(R⁰)C(O)OR, or —N(R⁰)C(O)N(R⁰)₂, wherein each R⁰        is independently hydrogen or C₁₋₆ alkyl.

The disclosure further comprises subgenera of formula (II) in which thesubstituents are selected as any and all combinations of one or more ofstructural formula (II), R¹, and R³, as defined herein, includingwithout limitation, the following:

Structural Formula II is One of Formulae (IIa)-(IIh):

R¹ is Selected from One of the Following Groups (6a)-(6ii):

-   -   (6a) R¹ is C₁₋₄ alkyl, —C₁₋₄ alkyl-R¹², C₂₋₄ alkynyl, C₃₋₈        cycloalkyl, monocyclic heterocyclyl, monocyclic heteroaryl, or        phenyl, wherein the cycloalkyl, heterocyclyl, heteroaryl, and        phenyl groups are each optionally substituted with one or two        R¹¹ groups.    -   (6b) R¹ is C₃₋₈ cycloalkyl, monocyclic heterocyclyl, monocyclic        heteroaryl, or phenyl, wherein the cycloalkyl, heterocyclyl,        heteroaryl, and phenyl groups are each optionally substituted        with one or two R¹¹ groups.    -   (6c) R¹ is C₃₋₈ cycloalkyl; or a monocyclic heterocyclyl        optionally substituted with one R¹¹ group.    -   (6d) R¹ is C₃₋₈ cycloalkyl.    -   (6e) R¹ is monocyclic heterocyclyl optionally substituted with        one R¹¹ group.    -   (6f) R¹ is piperidinyl or tetrahydropyranyl, each optionally        substituted with one R¹¹ group.    -   (6g) R¹ is phenyl optionally substituted with one or two R¹¹        groups.    -   (6h) R¹ is C₂₋₆ alkyl.    -   (6i) R¹ is C₁₋₄ alkyl.    -   (6j) R¹ is isopropyl or t-butyl.    -   (6k) R¹ is t-butyl.    -   (6l) R¹ is isopropyl.    -   (6m) R¹ is C₂₋₆ alkyl or —C₁₋₄ alkyl-R¹².    -   (6n) R¹ is —C₁₋₄ alkyl-R¹².    -   (6o) R¹ is —C₁₋₂ alkyl-R¹².    -   (6p) R¹ is —CH₂—R¹²    -   (6q) Any one of groups (6m)-(6p), wherein R¹² is —C(O)OR,        —C(O)NR₂, phenyl, monocyclic heteroaryl, C₃₋₈ cycloalkyl, or        monocyclic heterocyclyl, wherein the phenyl, heteroaryl, C₃₋₈        cycloalkyl, and heterocyclyl groups are each optionally        substituted by one, two, or three groups that are each        independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,        —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R,        —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂;    -   (6r) Any one of groups (6m)-(6p), R¹² is —C(O)OR or —C(O)NR₂.    -   (6s) Any one of groups (6m)-(6p), R¹² is phenyl, monocyclic        heteroaryl, C₃₋₈ cycloalkyl, or monocyclic heterocyclyl, each        optionally substituted by one, two, or three groups that are        each independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆        haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,        —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or        —N(R)C(O)NR₂.    -   (6t) Any one of groups (6m)-(6p), wherein R¹² is phenyl,        monocyclic heteroaryl, C₃₋₈ cycloalkyl, or monocyclic        heterocyclyl, wherein the phenyl, heteroaryl, C₃₋₈ cycloalkyl,        and heterocyclyl groups are each optionally substituted by one        or two groups that are each independently halogen, C₁₋₆ alkyl,        —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR,        —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂.    -   (6u) Any one of groups (6m)-(6p), R¹² is phenyl or monocyclic        heterocyclyl, each optionally substituted by one, two, or three        groups that are each independently halogen, cyano, nitro, C₁₋₆        alkyl, C₁₋₆ haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR,        —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R,        —N(R)C(O)OR, or —N(R)C(O)NR₂.    -   (6v) Any one of groups (6m)-(6p), R¹² is monocyclic heterocyclyl        optionally substituted by one, two, or three groups that are        each independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆        haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,        —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or        —N(R)C(O)NR₂.    -   (6w) Any one of groups (6m)-(6p), wherein R¹² is monocyclic        heterocyclyl optionally substituted by one or two groups that        are each independently halogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR,        —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R,        —N(R)C(O)OR, or —N(R)C(O)NR₂.    -   (6x) Any one of groups (6m)-(6p), R¹² is piperidinyl or        tetrahydropyranyl, each optionally substituted by one or two        groups that are each independently halogen, cyano, nitro, C₁₋₆        alkyl, C₁₋₆ haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR,        —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R,        —N(R)C(O)OR, or —N(R)C(O)NR₂.    -   (6y) Any one of groups (6m)-(6p), wherein R¹² is piperidinyl        optionally substituted by one or two groups that are each        independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,        —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R,        —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂.    -   (6z) Any one of groups (6m)-(6p), wherein R¹² is piperidinyl        optionally substituted by one or two groups that are each        independently C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A),        —C(O)N(R^(A))₂, —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A),        —OC(O)N(R^(A))₂, —N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or        —N(R^(A))C(O)N(R^(A))₂, wherein each R^(A) is independently        hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆ haloalkyl, C₃₋₈        cycloalkyl, heterocyclyl, aryl, arylC₁₋₆ alkyl, heteroaryl, or        heteroarylC₁₋₆ alkyl.    -   (6aa) Any one of groups (6m)-(6p), wherein R¹² is piperidinyl        optionally substituted by one or two groups that are each        independently C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,        —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or        —N(R)C(O)NR₂.    -   (6bb) Any one of groups (6m)-(6p), wherein R¹² is piperidinyl        optionally substituted by one or two groups that are each        independently C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A),        —C(O)N(R^(A))₂, —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A),        —OC(O)N(R^(A))₂, —N(R^(A)) C(O)R^(A), —N(R^(A))C(O)OR^(A), or        —N(R^(A))C(O)N(R^(A))₂, wherein each R^(A) is independently        hydrogen or C₁₋₆ alkyl.    -   (6cc) Any one of groups (6m)-(6p), wherein R¹² is piperidinyl        optionally substituted by one or two groups that are each        independently C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, or —S(O)₂R.    -   (6dd) Any one of groups (6m)-(6p), wherein R¹² is piperidinyl        optionally substituted by one or two groups that are each        independently C₁₋₆ alkyl, —C(O)R^(A), or —S(O)₂R^(A), wherein        each R^(A) is independently hydrogen or C₁₋₆ alkyl.    -   (6ee) Any one of groups (6m)-(6p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂,—S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or—N(R)C(O)NR₂.

-   -   (6ff) Any one of groups (6m)-(6p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A),—C(O)N(R^(A))₂, —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,—N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,wherein each R^(A) is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₁₋₆ haloalkyl, C₃₋₈ cycloalkyl, heterocyclyl, aryl, arylC₁₋₆ alkyl,heteroaryl, or heteroarylC₁₋₆ alkyl.

-   -   (6gg) Any one of groups (6m)-(6p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A),—C(O)N(R^(A))₂, —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,—N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,wherein each R^(A) is independently hydrogen or C₁₋₆ alkyl.

-   -   (6hh) Any one of groups (6m)-(6p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, or—S(O)₂R.

-   -   (6ii) Any one of groups (6m)-(6p), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), or —S(O)₂R^(A),wherein each R^(A) is independently hydrogen or C₁₋₆ alkyl.R³ is Selected from One of the Following Groups (7a)-(7x):

-   -   (7a) R³ is naphthyl, quinolinyl, pyridyl, pyrimidinyl, indolyl,        benzodioxolyl, 2,3-dihydrobenzodioxinyl, dibenzothienyl,        chromanyl, or phenyl, each optionally substituted with one, two,        or three R³¹ groups.    -   (7b) R³ is naphthyl, quinolinyl, indolyl, benzodioxolyl,        2,3-dihydrobenzodioxinyl, or chromanyl, each optionally        substituted with one, two, or three R³¹ groups.    -   (7c) R³ is quinolinyl, pyridyl, pyrimidinyl, or indolyl, each        optionally substituted with one, two, or three R³¹ groups.    -   (7d) R³ is quinolinyl or indolyl, each optionally substituted        with one, two, or three R³¹ groups.    -   (7e) R³ is quinolinyl optionally substituted with one, two, or        three R³¹ groups.    -   (7f) R³ is pyridyl or pyrimidinyl, each optionally substituted        with one, two, or three R³¹ groups.    -   (7g) R³ is phenyl substituted with one, two, or three R³¹        groups.    -   (7h) R³ is phenyl optionally substituted with one or two R³¹        groups, and substituted with one group that is halogen, C₁₋₆        alkyl, —OR, —SR, —NR₂, —N(R)C(O)R, or —N(R)S(O)₂R.    -   (7i) R³ is phenyl optionally substituted with one or two R³¹        groups, and substituted with one group that is —OR or        —N(R)C(O)R.    -   (7j) R³ is phenyl optionally substituted with one or two R³¹        groups, and substituted with one group that is —OR¹⁰, wherein        R¹⁰ is benzyl optionally substituted with one, two, three, or        four groups that are each independently halogen, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, or —OR.    -   (7k) R³ is phenyl optionally substituted with one or two R³¹        groups, and substituted with one group that is —N(H)C(O)R²⁰,        wherein R²⁰ is C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆ haloalkyl, aryl,        or arylC₁₋₆ alkyl.    -   (7l) R³ is naphthyl optionally substituted with one, two, or        three R³¹ groups.    -   (7m) R³ is naphthyl optionally substituted with one or two R³¹        groups and substituted with one group that is —OR¹⁵, wherein R¹⁵        is C₁₋₆ alkyl or benzyl, wherein the benzyl is optionally        substituted with one, two, three, or four groups that are each        independently halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, or —OR.    -   (7n) R³ is naphth-1-yl optionally substituted with one, two, or        three R³¹ groups.    -   (7o) R³ is naphth-1-yl optionally substituted with one or two        R³¹ groups and substituted with one group that is —OR¹⁵, wherein        R¹⁵ is C₁₋₆ alkyl or benzyl, wherein the benzyl is optionally        substituted with one, two, three, or four groups that are each        independently halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, or —OR⁰.    -   (7p) R³ is naphth-2-yl optionally substituted with one, two, or        three R³¹ groups.    -   (7q) R³ is naphth-2-yl optionally substituted with one or two        R³¹ groups and substituted with one group that is —OR¹⁵, wherein        R¹⁵ is C₁₋₆ alkyl or benzyl, wherein the benzyl is optionally        substituted with one, two, three, or four groups that are each        independently halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, or —OR⁰.    -   (7r) R³ is

-   -   (7s) R³ is indolyl optionally substituted with one, two, or        three R³¹ groups.    -   (7t) R³ is indol-2-yl optionally substituted with one, two, or        three R³¹ groups.    -   (7u) R³ is indol-3-yl optionally substituted with one, two, or        three R³¹ groups.    -   (7v) R³ is indol-5-yl optionally substituted with one, two, or        three R³¹ groups.    -   (7w) R³ is indol-6-yl optionally substituted with one, two, or        three R³¹ groups.    -   (7x) Any one of groups (2a)-(2aa) as defined above for formula        (I).

Other particular embodiments of this aspect of the invention includecompounds of any one of the formulae (II), (IIa)-(IIh), each as definedin each of the following rows, wherein each entry is a group number asdefined above (e.g., (7w) refers to R³ is indol-6-yl optionallysubstituted with one, two, or three R³¹ groups), and a dash “-”indicates that the variable is as defined for formula (II) or definedaccording to any one of the applicable variable definitions (6a)-(7x)[e.g., when R¹ is a dash, it can be either as defined for Formula (II)or any one of definitions (6a)-(6ii)]:

R¹ R³ (2)-1 6c 7a (2)-2 6c 7c (2)-3 6c 7e (2)-4 6c 7g (2)-5 6c 7k (2)-66c 7l (2)-7 6c 7m (2)-8 6c 7s (2)-9 6c 7t (2)-10 6c 7v (2)-11 6f 7a(2)-12 6f 7c (2)-13 6f 7e (2)-14 6f 7g (2)-15 6f 7k (2)-16 6f 7l (2)-176f 7m (2)-18 6f 7s (2)-19 6f 7t (2)-20 6f 7v (2)-21 6h 7a (2)-22 6h 7c(2)-23 6h 7e (2)-24 6h 7g (2)-25 6h 7k (2)-26 6h 7l (2)-27 6h 7m (2)-286h 7s (2)-29 6h 7t (2)-30 6h 7v (2)-31 6n 7a (2)-32 6n 7c (2)-33 6n 7e(2)-34 6n 7g (2)-35 6n 7k (2)-36 6n 7l (2)-37 6n 7m (2)-38 6n 7s (2)-396n 7t (2)-40 6n 7v (2)-41 6o 7a (2)-42 6o 7c (2)-43 6o 7e (2)-44 6o 7g(2)-45 6o 7k (2)-46 6o 7l (2)-47 6o 7m (2)-48 6o 7s (2)-49 6o 7t (2)-506o 7v (2)-51 6p 7a (2)-52 6p 7c (2)-53 6p 7e (2)-54 6p 7g (2)-55 6p 7k(2)-56 6p 7l (2)-57 6p 7m (2)-58 6p 7s (2)-59 6p 7t (2)-60 6p 7v (2)-616aa 7a (2)-62 6aa 7c (2)-63 6aa 7e (2)-64 6aa 7g (2)-65 6aa 7k (2)-666aa 7l (2)-67 6aa 7m (2)-68 6aa 7s (2)-69 6aa 7t (2)-70 6aa 7v

In another embodiment, the compound of formula (II) is one of compoundsof Table B,

TABLE B Cmpd # Structure Name  1

1-tert-butyl-3-(naphthalen-1-yl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine  2

1-tert-butyl-3-(naphthalen-2-yl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine  3

1-isopropyl-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5a

1-methyl-3-(naphthalen-1-ylmethyl)-1H- pyrazolo[3,4-d]pyrimidin-4-amine5b

3-(naphthalen-1-ylmethyl)-1-(prop-2-ynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5c

2-(4-amino-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)acetamide 5d

1-tert-butyl-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5e

1-benzyl-3-(naphthalen-1-ylmethyl)-1H- pyrazolo[3,4-d]pyrimidin-4-amine5f

1-cyclohexyl-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5g

3-(naphthalen-1-ylmethyl)-1-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5i

3-(naphthalen-1-ylmethyl)-1-(2-(piperidin-4-yl)ethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5h

3-(naphthalen-1-ylmethyl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5j

1-(4-(4-amino-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1- yl)ethanone 5p

1-(1-(methylsulfonyl)piperidin-4-yl)-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine 5m

1-(1-ethylpiperidin-4-yl)-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5k

1-(4-((4-amino-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)piperidin-1- yl)ethanone 5q

1-((1-(methylsulfonyl)piperidin-4-yl)methyl)-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine 5n

1-((1-ethylpiperidin-4-yl)methyl)-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 5l

1-(4-(2-(4-amino-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)piperidin-1- yl)ethanone 5r

1-(2-(1-(methylsulfonyl)piperidin-4-yl)ethyl)-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine 5o

1-(2-(1-ethylpiperidin-4-yl)ethyl)-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 7a

3-(4-chlorophenyl)-1-isopropyl-1H-pyrazolo[3,4- d]pyrimidin-4-amine 7b

3-(3-chlorophenyl)-1-isopropyl-1H-pyrazolo[3,4- d]pyrimidin-4-amine 7c

3-(3,4-dichlorophenyl)-1-isopropyl-1H- pyrazolo[3,4-d]pyrimidin-4-amine7d

1-isopropyl-3-(4-isopropylphenyl)-1H- pyrazolo[3,4-d]pyrimidin-4-amine7e

1-isopropyl-3-(3-isopropylphenyl)-1H- pyrazolo[3,4-d]pyrimidin-4-amine7f

1-isopropyl-3-p-tolyl-1H-pyrazolo[3,4- d]pyrimidin-4-amine 7g

1-isopropyl-3-m-tolyl-1H-pyrazolo[3,4- d]pyrimidin-4-amine 7h

3-(3,4-dimethylphenyl)-1-isopropyl-1H- pyrazolo[3,4-d]pyrimidin-4-amine7i

3-(4-fluoro-3-methylphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine 7j

1-isopropyl-3-(4-methoxyphenyl)-1H- pyrazolo[3,4-d]pyrimidin-4-amine 7k

3-(3,4-dimethoxyphenyl)-1-isopropyl-1H- pyrazolo[3,4-d]pyrimidin-4-amine7l

1-isopropyl-3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 7m

1-isopropyl-3-(4-methoxy-3-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 7n

1-(3-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)ethanone 7o

1-isopropyl-3-(naphthalen-1-yl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine 7p

1-isopropyl-3-(naphthalen-2-yl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine 7q

1-isopropyl-3-(6-methoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 7r

3-(6-ethoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine 7s

1-isopropyl-3-(3-methoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 7t

1-isopropyl-3-(quinolin-3-yl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine 7u

3-(benzo[d][1,3]dioxol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine 7v

3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine 7w

3-(chroman-6-yl)-1-isopropyl-1H-pyrazolo[3,4- d]pyrimidin-4-amine 7x

3-(dibenzo[b,d]thiophen-4-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine 7y

3-(3-benzylphenyl)-1-isopropyl-1H-pyrazolo[3,4- d]pyrimidin-4-amine 7z

3-(biphenyl-3-yl)-1-isopropyl-1H-pyrazolo[3,4- d]pyrimidin-4-amine 7aa

3-(3-(benzyloxy)phenyl)-1-isopropyl-1H- pyrazolo[3,4-d]pyrimidin-4-amine7ab

1-isopropyl-3-(3-(methylthio)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  8

3-isopropyl-1-(naphthalen-2-yl)imidazo[1,5- a]pyrazin-8-amine  9

3-isopropyl-1-(quinolin-3-yl)imidazo[1,5- a]pyrazin-8-amine  10

1-(6-ethoxynaphthalen-2-yl)-3- isopropylimidazo[1,5-a]pyrazin-8-amine 11

3-(6-isopropoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  12

1-isopropyl-3-(6-propoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  13

1-cyclopentyl-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  14

1-isopropyl-3-(naphthalen-2-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  15

3-(naphthalen-1-ylniethyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  16

1-isopropyl-3-(pyridin-3-yl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine  17

1-(cyclohexylmethyl)-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  18

1-isopropyl-3-(pyrimidin-5-yl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine  19

3-(2-chloropyridin-4-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  20

1-isopropyl-3-(1-methyl-1H-indol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  21

3-(3-(2-chlorobenzyloxy)phenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  22

3-(3-(3,5-dimethoxybenzyloxy)phenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  23

3-(3-isopropoxy-4-methoxyphenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  24

3-(1H-indol-2-yl)-1-isopropyl-1H-pyrazolo[3,4- d]pyrimidin-4-amine  25

1-isopropyl-3-(4-methyl-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 26

3-(4-chloro-1H-indol-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  27

1-isopropyl-3-(5-methoxy-1H-indol-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  28

3-(6-methoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  29

3-(6-ethoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  30

1-(piperidin-4-ylmethyl)-3-(quinolin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  31

1-((1H-imidazol-4-yl)methyl)-3-(naphthalen-1-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  32

3-(6-isopropoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  33

1-(piperidin-4-ylmethyl)-3-(6-propoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  34

3-(6-(benzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  35

3-(5-butoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  36

3-(6-(allyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  37

3-(6-(2-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine  38

3-(6-(3-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine  39

3-(6-(4-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine  40

3-(6-(benzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  41

3-(6-(allyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  42

3-(6-butoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  43

3-(6-isobutoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  44

3-(6-isobutoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  45

3-(4-chlorophenyl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  46

3-(6-(2-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  47

3-(6-(3-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  48

3-(6-(2,5-dimethylbenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  49

1-isopropyl-3-(6-(2-methylbenzyloxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  50

1-isopropyl-3-(6-(2-methyl-5-(trifluoromethyl)benzyloxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  51

3-(6-(3-chloro-4-(2,2,2- trifluoroethyl)benzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  52

3-(6-(3-chloro-5-fluorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4- ainine  53

1-isopropyl-3-(6-(1-phenylethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  54

3-(6-(4-tert-butylbenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  55

1-isopropyl-3-(6-(pyridin-4- ylmethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  56

3-(6-(4-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  57

3-(1-methyl-1H-indol-5-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  58

N-(4-(4-amino-1-tert-butyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)-2-chloroacetamide  59

N-(4-(4-amino-1-tert-butyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)isobutyramide  60

N-(4-(4-amino-1-tert-butyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)pivalamide  61

N-(4-(4-amino-1-tert-butyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)butyramide  62

N-(4-(4-amino-1-tert-butyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)-3-methylbutanamide  63

N-(4-(4-amino-1-tert-butyl-1H-pyrazolo[3,4- d]pyrimidin-3-yl)phenyl)cyclopentanecarboxamide  64

N-(4-(4-amino-1-tert-butyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)cyclohexanecarboxamide  65

N-(4-(4-amino-1-tert-butyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)benzamide  66

N-(4-(4-amino-1-tert-butyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)-2-phenylacetamide  67

N-(4-(4-amino-1-tert-butyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)phenyl)propionamide  68

6-(4-amino-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N,N- dimethylquinolin-2-amine  69

3-tert-butyl-1-(6-ethoxynaphthalen-2- yl)imidazo[1,5-a]pyrazin-8-amine 70

3-tert-butyl-1-(1-methyl-1H-indol-5- yl)imidazo[1,5-a]pyrazin-8-amine 71

3-tert-butyl-1-(6-methoxynaphthalen-2- yl)imidazo[1,5-a]pyrazin-8-amine 72

3-tert-butyl-1-(quinolin-3-yl)imidazo[1,5- a]pyrazin-8-amine  73

3-(2-fluorobiphenyl-4-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  74

4′-(4-amino-1-(piperidin-4-ylrnethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)biphenyl-3- carbonitrile  75

N-(4-(4-amino-1-phenyl-1H-pyrazolo[3,4- d]pyrimidin-3-yl)phenyl)-4-methylbenzenesulfonamide  76

3-(biphenyl-4-yl)-1-isopropyl-1H-pyrazolo[3,4- d]pyrimidin-4-amine  77

3-(naphthalen-1-ylmethyl)-1-(tetrahydrofuran-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine  78

3-(3-(benzyloxy)-2,6-difluorophenyl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine  79

1-allyl-3-(naphthalen-1-ylmethyl)-1H- pyrazolo[3,4-d]pyrimidin-4-amine 80

3-isopropyl-1-(6-methoxynaphthalen-2- yl)imidazo[1,5-a]pyrazin-8-amine147

3-(naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine 150

3-(6-ethoxynaphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4- d]pyrimidin-4-amine andpharmaceutically acceptable salts thereof.

In another embodiment, the bumped kinase inhibitor is of the formula(IV),

or a pharmaceutically acceptable salt thereof, wherein

-   X, Y, and Z are defined by either: (i) X is N, Y is C, and Z is N;    or (ii) X is C, Y is N, and Z is C(H);-   R¹ is C₁₋₆ alkyl, C₂₋₆ alkynyl, —C₁₋₆ alkyl-R¹², C₃₋₈ cycloalkyl,    monocyclic heterocyclyl, or phenyl, wherein    -   the monocyclic heterocyclyl and phenyl groups are each        optionally substituted with one or two R¹¹ groups;    -   each R¹¹ is independently C₁₋₆ alkyl, C₁₋₆ haloalkyl, —C(O)R, or        —S(O)₂R;    -   and    -   R¹² is —C(O)OR, —C(O)NR₂, phenyl, monocyclic heteroaryl, C₃₋₈        cycloalkyl, or monocyclic heterocyclyl, wherein the phenyl,        heteroaryl, C₃₋₈ cycloalkyl, and heterocyclyl groups are each        optionally substituted by one, two, or three groups that are        each independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆        haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,        —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or        —N(R)C(O)NR₂;-   L is a bond or —CH₂—;-   R³ is aryl or heteroaryl, each optionally substituted with one, two,    or three R³¹ groups,-   wherein    -   each R³¹ is independently halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl,        phenyl, benzyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂,        —OC(O)R, —N(R)C(O)R, or —N(R)S(O)₂R, wherein the phenyl and        benzyl groups are optionally substituted with one or two R¹⁰        groups;-   each R is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆    haloalkyl, C₃₋₈ cycloalkyl, aryl, arylC₁₋₆ alkyl, or heteroarylC₁₋₆    alkyl wherein the alkyl, aryl, arylalkyl, heteroaryl, and    heteroarylalkyl are optionally substituted with one or two R¹⁰    groups;-   and-   each R¹⁰ is independently halogen, cyano, C₁₋₆ alkyl, C₁₋₆    haloalkyl, —OR⁰, —SR⁰, or —N(R⁰)₂, wherein each R⁰ is independently    hydrogen or C₁₋₆ alkyl.

The disclosure further comprises subgenera of formula (IV) in which thesubstituents are selected as any and all combinations of one or more ofstructural formula (IV), R¹, and R³, as defined herein, includingwithout limitation, the following:

Structural Formula II is One of Formulae (IVa)-(IVh):

R¹ is Selected from One of the Following Groups (13a)-(13ff):

-   -   (13a) R¹ is C₃₋₈ cycloalkyl; or a monocyclic heterocyclyl        optionally substituted with one R¹ group.    -   (13b) R¹ is C₃₋₈ cycloalkyl.    -   (13c) R¹ is monocyclic heterocyclyl optionally substituted with        one R¹¹ group.    -   (13d) R¹ is piperidinyl or tetrahydropyranyl, each optionally        substituted with one R¹¹ group.    -   (13e) R¹ is phenyl optionally substituted with one or two R¹        groups.    -   (13f) R¹ is C₂₋₆ alkyl.    -   (13g) R¹ is C₁₋₄ alkyl.    -   (13h) R¹ is isopropyl or t-butyl.    -   (13i) R¹ is t-butyl.    -   (13j) R¹ is isopropyl.    -   (13k) R¹ is C₂₋₆ alkyl or —C₁₋₄ alkyl-R¹².    -   (13l) R¹ is —C₁₋₄ alkyl-R¹².    -   (13m) R¹ is —C₁₋₂ alkyl-R¹².    -   (13n) R¹ is —CH₂—R¹².    -   (13o) Any one of groups (13k)-(13n), R¹² is —C(O)OR or —C(O)NR₂.    -   (13p) Any one of groups (13k)-(13n), R¹² is phenyl, monocyclic        heteroaryl, C₃₋₈ cycloalkyl, or monocyclic heterocyclyl, each        optionally substituted by one, two, or three groups that are        each independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆        haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,        —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or        —N(R)C(O)NR₂.    -   (13q) Any one of groups (13k)-(13n), wherein R¹² is phenyl,        monocyclic heteroaryl, C₃₋₈ cycloalkyl, or monocyclic        heterocyclyl, wherein the phenyl, heteroaryl, C₃₋₈ cycloalkyl,        and heterocyclyl groups are each optionally substituted by one        or two groups that are each independently halogen, C₁₋₆ alkyl,        —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR,        —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂.    -   (13r) Any one of groups (13k)-(13n), R¹² is phenyl or monocyclic        heterocyclyl, each optionally substituted by one, two, or three        groups that are each independently halogen, cyano, nitro, C₁₋₆        alkyl, C₁₋₆ haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR,        —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R,        —N(R)C(O)OR, or —N(R)C(O)NR₂.    -   (13s) Any one of groups (13k)-(13n), R¹² is monocyclic        heterocyclyl optionally substituted by one, two, or three groups        that are each independently halogen, cyano, nitro, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂,        —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR,        or —N(R)C(O)NR₂.    -   (13t) Any one of groups (13k)-(13n), wherein R¹² is monocyclic        heterocyclyl optionally substituted by one or two groups that        are each independently halogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR,        —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R,        —N(R)C(O)OR, or —N(R)C(O)NR₂.    -   (13u) Any one of groups (13k)-(13n), R¹² is piperidinyl or        tetrahydropyranyl, each optionally substituted by one or two        groups that are each independently halogen, cyano, nitro, C₁₋₆        alkyl, C₁₋₆ haloalkyl, —OR, —SR, —NR₂, —C(O)R, —C(O)OR,        —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R,        —N(R)C(O)OR, or —N(R)C(O)NR₂.    -   (13v) Any one of groups (13k)-(13n), wherein R¹² is piperidinyl        optionally substituted by one or two groups that are each        independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,        —OR, —SR, —NR₂, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R,        —OC(O) OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂.    -   (13w) Any one of groups (13k)-(13n), wherein R¹² is piperidinyl        optionally substituted by one or two groups that are each        independently C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A),        —C(O)N(R^(A))₂, —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A),        —OC(O)N(R^(A))₂, —N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or        —N(R^(A))C(O)N(R^(A))₂, wherein each R^(A) is independently        hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆ haloalkyl, C₃₋₈        cycloalkyl, heterocyclyl, aryl, arylC₁₋₆ alkyl, heteroaryl, or        heteroarylC₁₋₆ alkyl.    -   (13x) Any one of groups (13k)-(13n), wherein R¹² is piperidinyl        optionally substituted by one or two groups that are each        independently C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,        —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or        —N(R)C(O)NR₂.    -   (13y) Any one of groups (13k)-(13n), wherein R¹² is piperidinyl        optionally substituted by one or two groups that are each        independently C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A),        —C(O)N(R^(A))₂, —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A),        —OC(O)N(R^(A))₂, —N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or        —N(R^(A))C(O)N(R^(A))₂, wherein each R^(A) is independently        hydrogen or C₁₋₆ alkyl.    -   (13z) Any one of groups (13k)-(13n), wherein R¹² is piperidinyl        optionally substituted by one or two groups that are each        independently C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, or —S(O)₂R.    -   (13aa) Any one of groups (13k)-(13n), wherein R¹² is piperidinyl        optionally substituted by one or two groups that are each        independently C₁₋₆ alkyl, —C(O)R^(A), or —S(O)₂R^(A), wherein        each R^(A) is independently hydrogen or C₁₋₆ alkyl.        (13bb) Any one of groups (13k)-(13n), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂,—S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or—N(R)C(O)NR₂.(13cc) Any one of groups (13k)-(13n), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A),—C(O)N(R^(A))₂, —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,—N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,wherein each R^(A) is independently hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl,C₁₋₆ haloalkyl, C₃₋₈ cycloalkyl, heterocyclyl, aryl, arylC₁₋₆ alkyl,heteroaryl, or heteroarylC₁₋₆ alkyl.(13dd) Any one of groups (13k)-(13n), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), —C(O)OR^(A),—C(O)N(R^(A))₂, —S(O)₂R^(A), —OC(O)R^(A), —OC(O)OR^(A), —OC(O)N(R^(A))₂,—N(R^(A))C(O)R^(A), —N(R^(A))C(O)OR^(A), or —N(R^(A))C(O)N(R^(A))₂,wherein each R^(A) is independently hydrogen or C₁₋₆ alkyl.

-   -   (13ee) Any one of groups (13k)-(13n), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, or—S(O)₂R.

-   -   (13ff) Any one of groups (13k)-(13n), wherein R¹² is

wherein R^(B) is hydrogen, C₁₋₆ alkyl, —C(O)R^(A), or —S(O)₂R^(A),wherein each R^(A) is independently hydrogen or C₁₋₆ alkyl.R³ is Selected from One of the Following Groups (14a)-(14x):

-   -   (14a) R³ is naphthyl, quinolinyl, pyridyl, pyrimidinyl, indolyl,        benzodioxolyl, 2,3-dihydrobenzodioxinyl, dibenzothienyl,        chromanyl, or phenyl, each optionally substituted with one, two,        or three R³¹ groups.    -   (14b) R³ is naphthyl, quinolinyl, indolyl, benzodioxolyl,        2,3-dihydrobenzodioxinyl, or chromanyl, each optionally        substituted with one, two, or three R³¹ groups.    -   (14c) R³ is quinolinyl, pyridyl, pyrimidinyl, or indolyl, each        optionally substituted with one, two, or three R³¹ groups.    -   (14d) R³ is quinolinyl or indolyl, each optionally substituted        with one, two, or three R³¹ groups.    -   (14e) R³ is quinolinyl optionally substituted with one, two, or        three R³¹ groups.    -   (14f) R³ is pyridyl or pyrimidinyl, each optionally substituted        with one, two, or three R³¹ groups.    -   (14g) R³ is phenyl substituted with one, two, or three R³¹        groups.    -   (14h) R³ is phenyl optionally substituted with one or two R³¹        groups, and substituted with one group that is halogen, C₁₋₆        alkyl, —OR, —SR, —NR₂, —N(R)C(O)R, or —N(R)S(O)₂R.    -   (14i) R³ is phenyl optionally substituted with one or two R³¹        groups, and substituted with one group that is —OR or        —N(R)C(O)R.    -   (14j) R³ is phenyl optionally substituted with one or two R³¹        groups, and substituted with one group that is —OR¹⁰, wherein        R¹⁰ is benzyl optionally substituted with one, two, three, or        four groups that are each independently halogen, C₁₋₆ alkyl,        C₁₋₆ haloalkyl, or —OR⁰.    -   (14k) R³ is phenyl optionally substituted with one or two R³¹        groups, and substituted with one group that is —N(H)C(O)R²⁰,        wherein R²⁰ is C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆ haloalkyl, aryl,        or arylC₁₋₆ alkyl.    -   (14l) R³ is naphthyl optionally substituted with one, two, or        three R³¹ groups.    -   (14m) R³ is naphthyl optionally substituted with one or two R³¹        groups and substituted with one group that is —OR¹⁵, wherein R¹⁵        is C₁₋₆ alkyl or benzyl, wherein the benzyl is optionally        substituted with one or two groups that are each independently        halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, or —OR⁰.    -   (14n) R³ is naphth-1-yl optionally substituted with one, two, or        three R³¹ groups.    -   (14o) R³ is naphth-1-yl optionally substituted with one or two        R³¹ groups and substituted with one group that is —OR¹⁵, wherein        R¹⁵ is C₁₋₆ alkyl or benzyl, wherein the benzyl is optionally        substituted with one or two groups that are each independently        halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, or —OR⁰.    -   (14p) R³ is naphth-2-yl optionally substituted with one, two, or        three R³¹ groups.    -   (14q) R³ is naphth-2-yl optionally substituted with one or two        R³¹ groups and substituted with one group that is —OR¹⁵, wherein        R¹⁵ is C₁₋₆ alkyl or benzyl, wherein the benzyl is optionally        substituted with one or two groups that are each independently        halogen, C₁₋₆ alkyl, C₁₋₆ haloalkyl, or —OR⁰.    -   (14r) R³ is

-   -   (14s) R³ is indolyl optionally substituted with one, two, or        three R³¹ groups.    -   (14t) R³ is indol-2-yl optionally substituted with one, two, or        three R³¹ groups.    -   (14u) R³ is indol-3-yl optionally substituted with one, two, or        three R³¹ groups.    -   (14v) R³ is indol-5-yl optionally substituted with one, two, or        three R³¹ groups.    -   (14w) R³ is indol-6-yl optionally substituted with one, two, or        three R³¹ groups.    -   (14x) Any one of groups (2a)-(2aa) as defined above for formula        (I).

Other particular embodiments of this aspect of the invention includecompounds of any one of the formulae (IV), (IVa)-(IVh), each as definedin each of the following rows, wherein each entry is a group number asdefined above (e.g., (14w) refers to R³ is indol-6-yl optionallysubstituted with one, two, or three R³¹ groups), and a dash “-”indicates that the variable is as defined for formula (IV) or definedaccording to any one of the applicable variable definitions (13a)-(14x)[e.g., when R¹ is a dash, it can be either as defined for Formula (IV)or any one of definitions (13a)-(13ff)]:

R¹ R³ (4)-1 13a 14b (4)-2 13a 14d (4)-3 13a 14e (4)-4 13a 14g (4)-5 13a14i (4)-6 13a 14k (4)-7 13a 14m (4)-8 13a 14q (4)-9 13a 14t (4)-10 13a14v (4)-11 13f 14b (4)-12 13f 14d (4)-13 13f 14e (4)-14 13f 14g (4)-1513f 14i (4)-16 13f 14k (4)-17 13f 14m (4)-18 13f 14q (4)-19 13f 14t(4)-20 13f 14v (4)-21 13l 14b (4)-22 13l 14d (4)-23 13l 14e (4)-24 13l14g (4)-25 13l 14i (4)-26 13l 14k (4)-27 13l 14m (4)-28 13l 14q (4)-2913l 14t (4)-30 13l 14v (4)-31 13v 14b (4)-32 13v 14d (4)-33 13v 14e(4)-34 13v 14g (4)-35 13v 14i (4)-36 13v 14k (4)-37 13v 14m (4)-38 13v14q (4)-39 13v 14t (4)-40 13v 14v (4)-41 13bb 14b (4)-42 13bb 14d (4)-4313bb 14e (4)-44 13bb 14g (4)-45 13bb 14i (4)-46 13bb 14k (4)-47 13bb 14m(4)-48 13bb 14q (4)-49 13bb 14t (4)-50 13bb 14v

Particular embodiments of this aspect of the invention include compoundsof any one of the formula (IVb) or (IVh), each as defined in each of thefollowing rows, wherein each entry is a group number as defined above(e.g., (13n) refers to R¹ is —CH₂—R¹²). In addition, any one of groups(13k)-(13n) can be either as defined for Formula (IV) or any one ofdefinitions (13p)-(13ff):

R¹ R³ (4)-51 13k 14a (4)-52 13k 14b (4)-53 13k 14l (4)-54 13k 14m (4)-5513k 14p (4)-56 13k 14q (4)-57 13k 14r (4)-58 13l 14a (4)-59 13l 14b(4)-60 13l 14l (4)-61 13l 14m (4)-62 13l 14p (4)-63 13l 14q (4)-64 13l14r (4)-65 13m 14a (4)-66 13m 14b (4)-67 13m 14l (4)-68 13m 14m (4)-6913m 14p (4)-70 13m 14q (4)-71 13m 14r (4)-72 13n 14a (4)-73 13n 14b(4)-74 13n 14l (4)-1 13n 14m (4)-2 13n 14p (4)-3 13n 14q (4)-1 13n 14r

In an embodiment of any of the preceding, the compound of formula (II)and (IV) is not one of the following compounds

One embodiment of the present disclosure provide a method of treating asubject in need of treatment for an apicomplexan-related diseasecomprising administering an effective amount of a compound of any offormulas (I)-(IV) or any embodiment thereof, that inhibits the activityof an apicomplexan calcium dependent protein kinase (CDPK).

Particular embodiments of the present disclosure provide a method oftreating cryptosporidiosis in a subject comprising administering aneffective amount of a compound of any of formulas (I)-(IV) or anyembodiment thereof, that inhibits the activity of Cryptosporidium parvumand C. hominus calcium dependent protein kinase 1 (CpCDPK1).

Other particular embodiments of the present disclosure provide a methodof treating cryptosporidiosis in a subject comprising administering aneffective amount of a compound of any of formulas (I)-(IV) or anyembodiment thereof, that inhibits the activity of T. gondii, calciumdependent protein kinase 1 (TgCDPK1).

Other particular embodiments of the present disclosure provide a methodof treating cryptosporidiosis in a subject comprising administering aneffective amount of a compound of any of formulas (I)-(IV) or anyembodiment thereof, that inhibits the activity of P. falciparum or P.berghei calcium dependent protein kinase 4 (PfCDPK4 or PbCDPK4).

Optionally, the compound of any one of formulas (I)-(IV) or anyembodiment thereof, can be administered in combination with a secondagent, such as agents specific for use against the specificapicomplexan-related disorder being treated.

In one embodiment, the apicomplexan protozoan related disease istoxoplasmosis. As understood by one of ordinary skill in the art,toxoplasmosis can encompass a number of pathologies, including, but notlimited to, encephalitis, retinitis, lymphadenopathy, disseminateddisease, and hepatitis. Toxoplasmosis infects most genera ofwarm-blooded animals, including humans, but the primary host is thefelid (cat) family.

Cats are the definitive host for the Toxoplasma organism. Infection withthis protozoan parasite is fairly common, but actual disease caused bythis parasite is relatively rare in cats. Cats can become infected byToxoplasma by eating any of the three infective stages of the parasites.The most common route of infection is probably by ingestion of tissuecysts in infected prey or in other raw meat. Toxoplasma multiply in thesmall intestines and in approximately two to three weeks the oocysts areexcreted in the infected cat's feces. In another example, cats may betreated prophylactically for toxoplasmosis (e.g, a gastrointestinalinfection) provided by providing a therapeutically effective amount of acompounds of any one of formulas (I)-(IV) or to eliminate the chancethat they would shed infectious Toxoplasmodia oocyts and infect theirowners. In another embodiment, infected cats may be treated by providinga therapeutically effective amount of a compound of any one of formulas(I)-(IV) to treat toxoplasmosis. As will be understood by those of skillin the art, similar prophylactic and therapeutic methods for limitingdevelopment of or treating toxoplasmosis can be used in any animal thatcan be infected by Toxoplasma sp.

Animals are infected by eating infected meat, by ingestion of feces of acat that has itself recently been infected, or by transmission frommother to fetus. While cats are often blamed for spreadingtoxoplasmosis, contact with raw meat is a more significant source ofhuman infections in many countries, and fecal contamination of hands isa greater risk factor. Infection has two stages (1) acute toxoplasmosis;and (2) latent toxoplasmosis. During acute toxoplasmosis, symptoms areoften influenza-like: swollen lymph nodes, or muscle aches and painsthat last for a month or more. Rarely, a patient with a fullyfunctioning immune system may develop eye damage from toxoplasmosis.Young children (15 years old or younger) and immunocompromised patients,such as those with HIV/AIDS, those taking certain types of chemotherapy,or those who have recently received an organ transplant, may developsevere toxoplasmosis. In an embodiment, a young child can be 14 yearsold or younger; or 13 years old or younger; or 12 years old or younger;or 11 years old or younger; or 10 years old or younger; or 9 years oldor younger; or 8 years old or younger; or 7 years old or younger; or 6years old or younger; or 5 years old or younger; or 4 years old oryounger; or 3 years old or younger; or 2 years old or younger; or 1 yearold or younger. This can cause damage to the brain (encephalitis) or theeyes (necrotizing retinochoroiditis). Infants infected via placentaltransmission may be born with either of these problems, or with nasalmalformations, although these complications are rare in newborns. Inmost immunocompetent patients, the infection enters a latent phase,during which only bradyzoites are present, forming cysts in nervous andmuscle tissue. Most infants who are infected while in the womb have nosymptoms at birth but may develop symptoms later in life. The mostcommon current therapy for toxoplasmosis is sulfadiazine/pyrimethaminecombination therapy, but therapy is often limited by allergic reactionsto the sulfa component, anemia and pancytopenia induced by blocking thefolate pathway. When sulfadiazine cannot be used, clindamycin may becombined with pyrimethamine but most experts feel it does not work aswell as sulfadiazine. Spiramycin has been used for toxoplasmosis duringpregnancy but has issues with low efficacy and is no longer available inthe United States. Thus few therapeutic alternatives are available.

In another embodiment, the apicomplexan protozoan related disease iscryptosporidiosis. Cryptosporidiosis is caused by infection with thesingle-celled parasite (not bacterium) Cryptosporidium parvum. Thisparasite is found in many mammals including lambs, calves, goat kids,piglets and humans. Research so far has shown two basic types, thebovine type which affects most species, and a second human type whichcauses disease in humans only. Outbreaks of human disease, where largenumbers of people are affected, are usually water-borne and usuallyassociated with the bovine type of cryptosporidium. Individual sporadiccases of cryptosporidiosis in humans are mostly (around 60%) associatedwith the human type of cryptosporidium.

Cryptosporidiosis affects the intestines of mammals and is typically anacute short-term infection. It is spread through the fecal-oral route,often through contaminated water; the main symptom is self-limitingdiarrhea in people with intact immune systems. In immunocompromisedindividuals, such as HIV/AIDS patients, the symptoms are particularlysevere and often fatal. Cryptosporidium is the organism most commonlyisolated in HIV positive patients presenting with diarrhea.Cryptosporidiosis is one of the most common waterborne diseases and isfound worldwide. The parasite is transmitted by environmentally hardymicrobial cysts (oocysts) that, once ingested, exist in the smallintestine and result in an infection of intestinal epithelial tissue.Infection is through contaminated material such as earth, water,uncooked or cross-contaminated food that has been in contact with thefeces of an infected individual or animal. It is especially prevalentamongst those in regular contact with bodies of fresh water includingrecreational water such as swimming pools. Other potential sourcesinclude insufficiently treated or insufficiently filtered watersupplies, contaminated food, or exposure to feces. Symptoms appear fromtwo to ten days after infection, and last for up to two weeks or more.In immunocompetent people, the disease can be asymptomatic or causeacute diarrhea or persistent diarrhea that can last for a few weeks.There is often stomach pain or cramping and a low fever. Other symptomsmay include nausea, vomiting, malabsorption, and dehydration.Individuals who are asymptomatic (have no symptoms) are neverthelessinfective. Immunocompromised people, as well as very young or very oldpeople, can develop a more severe form of cryptosporidiosis. WhenCryptosporidium spreads beyond the intestine, as it can predominantly inpatients with AIDS, it can reach the lungs, middle ear, pancreas, andstomach. Thus, one symptom is pain in the right upper quadrant. Theparasite can infect the biliary tract, causing biliarycryptosporidiosis. This can result in cholecystitis and cholangitis.Current treatment is symptomatic, with fluid rehydration, electrolytecorrection and management of any pain.

Nitazoxanide has been FDA-approved for treatment of diarrhea caused byCryptosporidium in people with healthy immune systems and is availableby prescription, however it only shortens the duration of diarrhea by acouple of days. The effectiveness of nitazoxanide in immunosuppressedindividuals is unclear and multiple trials have shown no benefit.

The inhibitors described herein may have use in other apicoplexaprotozoan related diseases, such as coccidiosis caused by Eimeria spp.,cause infections and disease in poultry; which causes Babesiosis whichis caused by Babesia spp. and results in a malaria-like disease, andmalaria in humans and animals caused by Plasmodium spp.

Plasmodium calcium dependent protein kinase 4 (CDPK4) is essential forexflaggelation of microgametes, sexual reproduction and infection of themosquito host and is a potential drug target to block mosquitotransmission. Plasmodium transmission-blocking compounds that act viainhibition of PfCDPK4 have great promise in the armamentarium of malariacontrol. Plasmodium CDPK4 has a unique ATP binding site which rendersCDPK4 differentially sensitive to bumped kinase inhibitors (BKIs).TgCDPK1 and CpCDPK1 have ATP-binding pockets with an atypically smallgatekeeper residue, glycine. P. falciparum CDPK4 (PfCDPK4) has a serineresidue at the gatekeeper position, smaller than any gatekeeper inmammalian kinases, and an overall ATP-binding pocket that is verysimilar to TgCDPK1 and CpCDPK1. BKIs inhibit P. falciparum CDPK4(PfCDPK4) and prevents the exflagellation of malaria microgametes.Administration of BKIs to mice stops the transmission of P. berghei tomosquitoes. Finally, addition of BKIs to blood containing P. falciparumgametocytes stops exflagellation of microgametocytes and blocks theinfection of mosquitoes. BKIs are non-toxic, selective inhibitors thatblock malaria transmission to mosquitos, have favorable oralpharmacokinetic (PK) properties, have a low likelihood of generatingresistance.

Thus, other particular embodiments of the present disclosure provide amethod for treating malaria comprising administering an effective amountof a compound of any of formulas (I)-(IV) or any embodiment thereof,that inhibits the activity of Plasmodium falciparum and P. bergheicalcium dependent protein kinases. In one embodiment, the compound canbe administered in combination with a second agent. In anotherembodiment, the subject has malaria, and the second agent is ananti-malarial therapeutic. The subject can be human. In furtherembodiments, the subject is a mammal other than a human, such as a cator livestock (e.g., pigs, sheep, goats, cattle).

As used herein, the term “subject”, “individual,” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, birds, swine, horses,livestock (e.g., pigs, sheep, goats, cattle), primates or humans.

As used here, a subject “in need thereof” refers to a subject that hasthe disorder or disease to be treated or is predisposed to or otherwiseat risk of developing the disease or disorder.

As used here, the terms “treatment” and “treating” means

-   -   (i) inhibiting the progression of the disease;    -   (ii) prophylactic use for example, preventing or limiting        development of a disease, condition or disorder in an individual        who may be predisposed or otherwise at risk to the disease,        condition or disorder but does not yet experience or display the        pathology or symptomatology of the disease;    -   (iii) inhibiting the disease; for example, inhibiting a disease,        condition or disorder in an individual who is experiencing or        displaying the pathology or symptomatology of the disease,        condition or disorder;    -   (iv) ameliorating the referenced disease state, for example,        ameliorating a disease, condition or disorder in an individual        who is experiencing or displaying the pathology or        symptomatology of the disease, condition or disorder (i.e.,        reversing or improving the pathology and/or symptomatology) such        as decreasing the severity of disease;    -   (v) eliciting the referenced biological effect, or    -   (vi) limiting transmission of the disease.

In various preferred embodiments, the individual may beimmunocompromised (having an immune system that has been impaired bydisease or treatment, such as an HIV infected patient, and AIDS patient,or a patient receiving chemotherapy or an organ transplant), a pregnantfemale, fifteen years old or younger, fifty-five years old or older,exposed to contaminated water supplies, and/or exposed to other sourcesof contamination (fecal matter, blood transfusion, earth, food, etc.)The methods may further comprise administering the compounds disclosedherein to subjects at risk of acquiring an apicomplexan-related disease,such as those with compromised immune systems or that are extremelyyoung in high risk areas.

In other embodiments, the compounds described herein can be used inprophylactic manner. Cryptosporidiosis is usually seen in calves betweenone and two weeks of age and presents with diarrhea, colic and pain,depression, loss of appetite, and weight loss. Thus, in one embodiment,calves may be treated prophylactically by providing an effective amountof a compound of any one of formulas (I)-(IV) to limit the contractionor transmission of cryptosporidiosis. In a preferred embodiment, theadministering is done within the first 7-8 days after birth (day 1, 2,3, 4, 5, 6, 7, or 8) when calves are most susceptible to Cryptosporidiainfection. Such treatments may be repeated as necessary as would beunderstood by one skilled in the art.

In another embodiment, infected cattle may be treated by providing atherapeutically effective amount of a compound of any one of formulas(I)-(IV) to treat cryptosporidiosis. Such treatments may be repeated asnecessary as would be understood by one skilled in the art. In thisembodiment, the compounds of the invention may be administered togetherwith electrolytes if cattle become dehydrated. If disease is severe,halfuginone can be used in combination with the compounds of theinvention to reduce disease severity and prevent spread to otheranimals.

In another example, lambs are susceptible to cryptosporidiosis and maybe provided a therapeutically effective amount of a compounds of any oneof formulas (I)-(IV) to limit the contraction or transmission ofcryptosporidiosis.

In another example, any or all members of a herd (e.g., cattle, goats,lambs, etc.), may be provided a therapeutically effective amount of acompound of any one of formulas (I)-(IV) to limit the contraction ortransmission of toxoplasmosis or cryptosporidiosis or to rid the herd ofcattle of toxoplasmosis or cryptosporidiosis.

In another embodiment, goat kids may be treated prophylactically byproviding an effective amount of a compound of any one of formulas(I)-(IV) to limit the contraction or transmission of cryptosporidiosis.In a preferred embodiment, the administering is done within the first7-8 days after birth (day 1, 2, 3, 4, 5, 6, 7, or 8) when kids are mostsusceptible to cryptosporidiosis. Such treatments may be repeated asnecessary as would be understood by one skilled in the art. The extentto which a kid is infected seems to be dependent on its age and immunestatus. Younger animals are much more susceptible to infection thanadults. In studies done with lambs, five-day-old lambs had diarrhea for9-10 days and suffered from a high rate of mortality. Sixty-day-oldlambs showed no symptoms when they were infected, and adult sheepcompletely resisted infection. There is an indication that adultsdevelop an immunity to Cryptosporidium, yet this immunity does not seemto be passed to their offspring.

Immune-depressed goats are very susceptible to the disease. This refersto the total immune status, not just protection from cryptosporidiosis.Many situations can cause animals to lack immunity. Animals with severeinfections are more susceptible to secondary infections. The most commonproblem with kids is receiving a deficient amount of colostralantibodies following birth. Whether caused by disease, an imbalancedration or improper management, animals lacking adequate immunity aremuch more susceptible to cryptosporidiosis.

In another embodiment, infected goats may be treated by providing atherapeutically effective amount of a compound of any one of formulas(I)-(IV) to treat cryptosporidiosis. In a preferred embodiment, the goatis a kid.

In another example, pigs are susceptible to cryptosporidiosis and may beprovided a therapeutically effective amount of a compound of any one offormulas (I)-(IV) to limit the contraction or transmission ofcryptosporidiosis. In a preferred embodiment, the administering is donewithin the first 21 days after birth (day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21) when piglets are mostsusceptible to cryptosporidiosis and/or most likely

In another embodiment, infected pigs may be treated by providing atherapeutically effective amount of a compound of any one of formulas(I)-(IV) to treat cryptosporidiosis. In a preferred embodiment, the pigis a piglet.

In another example, birds, such as turkeys and chickens, are susceptibleto cryptosporidiosis and may be provided an effective amount of acompounds of any one of formulas (I)-(IV) to prevent the contraction ortransmission of cryptosporidiosis. In particular, Cryptosporidiumbaileyi can cause respiratory disease in chickens and turkeys. The samespecies causes infections of the hindgut and cloacal bursa in chickens,turkeys, and ducks. C. meleagridis also infects both species. A furtherspecies causes respiratory disease in quail. The oocysts are excretedready sporulated in the faeces and infection occurs by inhalation andingestion. Signs of cryptosporidiosis in poultry include snick, cough,swollen sinuses, low weight gain, and diarrhea. In another embodiment,infected birds may be treated by providing a therapeutically effectiveamount of a compound of any one of formulas (I)-(IV) to treatcryptosporidiosis.

In another example, birds, such as turkeys and chickens, are susceptibleto coccidiosis due to Eimeria infections and may be provided aneffective amount of a compounds of any one of formulas (I)-(IV) to limitthe contraction or transmission of coccidiosis, a parasitic diseasecaused by the development and multiplication of coccidia in theepithelial cells of the intestine. Eimeria infections are ubiquitous;they are found wherever chickens or turkeys are reared (traditional,industrial, label or organic/bio farms). Particular strains of Eimeriaknown to infect birds include, but are not limited to, Eimeriaacervulina. Eimeria adenoeides, Eimeria brunette, Eimeria colchici,Eimeria curvata, Eimeria dispersa, Eimeria duodenali, Eimeriafraterculae, Eimeria gallopavonis, Eimeria praecox, Eimeria maxima,Eimeria meleagrimitis, Eimeria mitis, Eimeria necatrix, Eimeriaphasiani, Eimeria procera, and Eimeria tenella. In another embodiment,infected birds may be treated by providing a therapeutically effectiveamount of a compound of any one of formulas (I)-(IV) to treatcoccidiosis

In another example, mammals, such as goats, sheep, llamas, alpacas,cattle, rabbits, and mice, are susceptible to coccidiosis and may beprovided an effective amount of a compounds of any one of formulas(I)-(IV) to limit the contraction or spreading of Eimeria. Particularstrains of Eimeria known to infect mammals include, but are not limitedto, Eimeria ahsata, Eimeria alabamensis, Eimeria alijevi, Eimeriaapsheronica, Eimeria arloingi, Eimeria arundeli, Eimeria bakuensis,Eimeria bovis, Eimeria cameli, Eimeria caprina, Eimeria caprovina,Eimeria christenseni, Eimeria clethrionomyis, Eimeria coecicola, Eimeriacontorta, Eimeria couesii, Eimeria crandallis, Eimeria dammahensis,Eimeria dowleri, Eimeria exigua, Eimeria falciformis, Eimeria farasanii,Eimeria ferrisi, Eimeria flavescens, Eimeria gallatii, Eimeriagranulosa, Eimeria hirci, Eimeria intestinalis, Eimeria irresidua,Eimeria intricata, Eimeria jolchijevi, Eimeria krijgsmanni, Eimerialarimerensis, Eimeria macusaniensis, Eimeria magna, Eimeria marconii,Eimeria media, Eimeria melanuri, Eimeria myoxi, Eimeria nagpurensis,Eimeria ninakohlyakimovae, Eimeria ovinoidalis, Eimeria pallida, Eimeriapalustris, Eimeria papillata, Eimeria perforans, Eimeria phocae, Eimeriapileata, Eimeria pipistrellu, Eimeria piriformis, Eimeria prionotemni,Eimeria punctate, Eimeria roobroucki, Eimeria saudiensis, Eimeriasealanderi, Eimeria separate, Eimeria stiedae, Eimeria ursini, Eimeriavermiformis, Eimeria weybridgensis, Eimeria wobati, and Eimeria zuernii.In another embodiment, infected mammals may be treated by providing atherapeutically effective amount of a compound of any one of formulas(I)-(IV) to treat coccidiosis

The usual age range for animals suffering from coccidiosis is from threeweeks to one year of age, but cattle remain susceptible to coccidiosisthroughout their lives or until they develop acquired immunity. Thesusceptibility of the animals is influenced by nutritional status(colostrum supply), stress (overstocking, transport, climate, hygiene,etc.), immune status and the occurrence of concurrent diseases.

Infections with multiple Eimeria species (pathogenic and non-pathogenic)are common in real life situations. The most important species relatedto the clinical manifestation of the disease in the stable are Eimeriabovis and Eimeria zuernii, although other pathogenic coccidia speciesmay also affect the cattle in the stables, such as Eimeria alabamensis(animals fed on contaminated hay), which is commonly associated withdiarrheic problems in animals that are released to pasture.

“Carrier hosts” shed relatively fewer oocysts and the susceptible“multiplier hosts” pick up the infection and shed many-fold oocysts intothe environment. Exposure to multiplier hosts leads to subclinical ormildly clinical infection in animals exposed to a large number ofoocysts in the environment. Calves exposed to a large number of oocystsare likely to develop severe coccidiosis. In feedlots where few oocystsare present, stress factors such as weaning, diet, temperature extremesand other variables may make the calves more susceptible to infectionand under such conditions the reproductive potential of coccidia in thegut greatly increases.

In goats, although the infection can occur in any goat herd raised undersemi and intensive management practices, it is most frequently observedin kids 2 to 4 weeks postweaning. The infection occurs by ingesting thepathogenic sporulated oocyst (sporulated is a form of resistance of theCoccidia). Oocysts can be found in the water or in feed suppliescontaminated with feces. Once ingested, oocysts penetrate the cellslining the intestine where they go through several stages of developmentand cause inflammation and destruction of intestinal cells. Stress isthe predisposing factor in kids during the postweaning period. Outbreakscan occur during stressful conditions such as after shipping or whenanimals are relocated. Outbreaks can also occur during sudden weatherchanges, after a change in concentrated feed practices, when animals arerecovering from a disease, or in worm burden cases. Although coccidiosiscan occur year around, a higher incidence occurs during postweaning.

The compounds disclosed herein can be used to treat coccidiosis incombination with standard treatments such as, but not limited to,replacing fluids by administering liquid nutritional supplement orallyby nipple bottle until the animal is rehydrated. Animals that have lost5 percent of their body weight may require intravenous (IV) and/orelectrolyte therapy. Treatment may include IV or subcutaneously (SC)fluid therapy with a physiologically balanced electrolyte such asRinger's, Plasmalyte-A, or Normosol-R. Administer the solution (2 to 5milliliters per pound) one to three times daily until the animal isrehydrated. Sulfas such as Albon™, Sulmet™, or Di-Methox™, can also bemixed in the drinking water or as a drench for individual goats. Analternative is CORID™ (amprolium).

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician, which includes one or more of thefollowing: (1) preventing the disease; for example, preventing adisease, condition or disorder in an individual who may be predisposedto the disease, condition or disorder but does not yet experience ordisplay the pathology or symptomatology of the disease; (2) inhibitingthe disease; for example, inhibiting a disease, condition or disorder inan individual who is experiencing or displaying the pathology orsymptomatology of the disease, condition or disorder; and (3)ameliorating the disease; for example, ameliorating a disease, conditionor disorder in an individual who is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder (i.e.,reversing the pathology and/or symptomatology) such as decreasing theseverity of disease.

Methods for Identifying a Compound

As disclosed herein, the inventors have identified compounds for use intreating apicomplexan-related disorders. While not being bound by aspecific mechanism of action, the inventors believe that the inhibitoryactivity is based on selective inhibition of apicomplexan proteinkinases, specifically selective inhibitors against apicomplexancalcium-dependent protein kinases (CDPKs). The inventors have furtheridentified a specific region of such CDPKs that the inhibitors bind to,exemplified by SEQ ID NO:1 (from the T. gondii, CDPK (SEQ ID NO: 13);(TgCDPK))) and SEQ ID NO:2 (from the C. parvum CDPK (CpCDPK)(SEQ ID NOs:14, 15, 16, 17)). Thus, another aspect of the present disclosureprovides a method of identifying a compound for treating anapicomplexan-related disease, including but not limited to thosedisclosed above, and most preferably toxoplasmosis andcryptosporidiosis. The method comprises (a) contacting a polypeptidecomprising an amino acid sequence according to SEQ ID NO. 1 or SEQ IDNO:2 with a compound to be tested for the ability to bind to thepolypeptide under conditions suitable for binding to the polypeptide,and (b) identifying compounds that bind to the polypeptide. Suitableconditions for such binding can be determined by one of skill in the artbased on the teachings herein. For example, the methods may comprisesuitable wash steps to remove unbound compounds. The methods may beconducted in vitro, or the binding may be assayed in cells usingappropriate reporter molecules.

In one embodiment, the method comprises contacting the compound with therecited peptide. In another embodiment, the compound is contacted withTgCDPK or CpCDPK. In a further embodiment, the compound is contactedwith TgCDPK or CpCDPK under conditions for crystallizing the complex, asdescribed in the examples that follow. In all of these embodiments, themethod may further comprise comparing binding of the compound to acontrol. Any suitable control can be used. In one embodiment, thecontrol comprises comparing binding of the polypeptide to binding of thecompound to a control polypeptide. Any suitable control polypeptide maybe used. In one embodiment, the control polypeptide may comprise amutated CDPK active region, such as TgCDPK1 G128M (described below), orcorresponding C. parvum CDPK mutated at the gatekeeper glycine residue(CpCDPK residue 150) (as described in detail herein). For example, thegatekeeper glycine residue in any TgCDPK or CpCDPK may be mutated asdescribed herein to replace the glycine residue with alanine, arginine,asparagine, aspartic acid, cysteine, glutamic acid, glutamine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, or valine. In theseembodiments, those compounds that show selective binding to thepolypeptide and little to no binding to the control peptide areidentified as selective inhibitors against apicomplexan CDPKs. Exemplarymethods according to this aspect of the invention are described in theexamples that follow.

In a further embodiment, the methods may comprise a verification step(c), comprising applying a compound selected in (b) to the relevantapicomplexan species, such Toxoplasma gondii of C. parvum, to test forinhibitory activity. Exemplary such assays for inhibitory activity aredescribed in the examples herein.

When the test compounds comprise polypeptide sequences, suchpolypeptides may be chemically synthesized or recombinantly expressed.Recombinant expression can be accomplished using standard methods in theart, as disclosed above. Such expression vectors can comprise bacterialor viral expression vectors, and such host cells can be prokaryotic oreukaryotic. Synthetic polypeptides, prepared using the well-knowntechniques of solid phase, liquid phase, or peptide condensationtechniques, or any combination thereof, can include natural andunnatural amino acids. Amino acids used for peptide synthesis may bestandard Boc (Nα-amino protected Nα-t-butyloxycarbonyl) amino acid resinwith standard deprotecting, neutralization, coupling and wash protocols,or standard base-labile Nα-amino protected 9-fluorenylmethoxycarbonyl(Fmoc) amino acids. Both Fmoc and Boc Nα-amino protected amino acids canbe obtained from Sigma, Cambridge Research Biochemical, or otherchemical companies familiar to those skilled in the art. In addition,the polypeptides can be synthesized with other Nα-protecting groups thatare familiar to those skilled in this art. Solid phase peptide synthesismay be accomplished by techniques familiar to those in the art andprovided, such as by using automated synthesizers.

When the test compounds comprise antibodies, such antibodies can bepolyclonal or monoclonal. The antibodies can be humanized, fully human,or murine forms of the antibodies. Such antibodies can be made bywell-known methods, such as described in Harlow and Lane, Antibodies; ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1988).

When the test compounds comprise nucleic acid sequences, such nucleicacids may be chemically synthesized or recombinantly expressed as well.Recombinant expression techniques are well known to those in the art(See, for example, Sambrook, et al., 1989, supra). The nucleic acids maybe DNA or RNA, and may be single stranded or double. Similarly, suchnucleic acids can be chemically or enzymatically synthesized by manualor automated reactions, using standard techniques in the art. Ifsynthesized chemically or by in vitro enzymatic synthesis, the nucleicacid may be purified prior to introduction into the cell. For example,the nucleic acids can be purified from a mixture by extraction with asolvent or resin, precipitation, electrophoresis, chromatography, or acombination thereof. Alternatively, the nucleic acids may be used withno or a minimum of purification to avoid losses due to sampleprocessing.

When the test compounds comprise compounds other than polypeptides,antibodies, or nucleic acids, such compounds can be made by any of thevariety of methods in the art for conducting organic chemical synthesis.

Pharmaceutical Compositions

The pharmaceutical compositions described herein generally comprise acombination of a compound described herein and a pharmaceuticallyacceptable carrier, diluent, or excipient. Such compositions aresubstantially free of non-pharmaceutically acceptable components, i.e.,contain amounts of non-pharmaceutically acceptable components lower thanpermitted by US regulatory requirements at the time of filing thisapplication. In some embodiments of this aspect, if the compound isdissolved or suspended in water, the composition further optionallycomprises an additional pharmaceutically acceptable carrier, diluent, orexcipient. In other embodiments, the pharmaceutical compositionsdescribed herein are solid pharmaceutical compositions (e.g., tablet,capsules, etc.).

These compositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration may be topical (includingophthalmic and to mucous membranes including intranasal, vaginal andrectal delivery), pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), ocular, oral or parenteral. Methods forocular delivery can include topical administration (eye drops),subconjunctival, periocular or intravitreal injection or introduction byballoon catheter or ophthalmic inserts surgically placed in theconjunctival sac. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Parenteral administration can be in the form of a singlebolus dose, or may be, for example, by a continuous perfusion pump.Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Also, pharmaceutical compositions can contain, as the active ingredient,one or more of the compounds described herein above in combination withone or more pharmaceutically acceptable carriers. In making thecompositions described herein, the active ingredient is typically mixedwith an excipient, diluted by an excipient or enclosed within such acarrier in the form of, for example, a capsule, sachet, paper, or othercontainer. When the excipient serves as a diluent, it can be a solid,semi-solid, or liquid material, which acts as a vehicle, carrier ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, syrups, aerosols (as a solid or in aliquid medium), ointments containing, for example, up to 10% by weightof the active compound, soft and hard gelatin capsules, suppositories,sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g. about 40 mesh.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions described herein can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 100 mg, more usually about 10 to about30 mg, of the active ingredient. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient.

The active compound can be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound described herein. When referring to these preformulationcompositions as homogeneous, the active ingredient is typicallydispersed evenly throughout the composition so that the composition canbe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation is thensubdivided into unit dosage forms of the type described above containingfrom, for example, 0.1 to about 500 mg of the active ingredient of acompound described herein.

The tablets or pills can be coated or otherwise compounded to provide adosage form affording the advantage of prolonged action. For example,the tablet or pill can comprise an inner dosage and an outer dosagecomponent, the latter being in the form of an envelope over the former.The two components can be separated by an enteric layer which serves toresist disintegration in the stomach and permit the inner component topass intact into the duodenum or to be delayed in release. A variety ofmaterials can be used for such enteric layers or coatings, suchmaterials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol, andcellulose acetate.

The liquid forms in which the compounds and compositions can beincorporated for administration orally or by injection include aqueoussolutions, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in can be nebulized by use of inert gases. Nebulizedsolutions may be breathed directly from the nebulizing device or thenebulizing device can be attached to a face masks tent, or intermittentpositive pressure breathing machine. Solution, suspension, or powdercompositions can be administered orally or nasally from devices whichdeliver the formulation in an appropriate manner.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of the compounds can vary according to, forexample, the particular use for which the treatment is made, the mannerof administration of the compound, the health and condition of thepatient, and the judgment of the prescribing physician. The proportionor concentration of a compound described herein in a pharmaceuticalcomposition can vary depending upon a number of factors includingdosage, chemical characteristics (e.g., hydrophobicity), and the routeof administration. For example, the compounds described herein can beprovided in an aqueous physiological buffer solution containing about0.1 to about 10% w/v of the compound for parenteral administration. Sometypical dose ranges are from about 1 μg/kg to about 1 g/kg of bodyweight per day. In some embodiments, the dose range is from about 0.01mg/kg to about 100 mg/kg of body weight per day. The dosage is likely todepend on such variables as the type and extent of progression of thedisease or disorder, the overall health status of the particularpatient, the relative biological efficacy of the compound selected,formulation of the excipient, and its route of administration. Effectivedoses can be extrapolated from dose-response curves derived from invitro or animal model test systems.

The compounds described herein can also be formulated in combinationwith one or more additional active ingredients which can include anypharmaceutical agent such as anti-viral agents, vaccines, antibodies,immune enhancers, immune suppressants, anti-inflammatory agents and thelike.

Definitions

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words ‘comprise’, ‘comprising’, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”. Words using the singular or pluralnumber also include the plural or singular number, respectively.Additionally, the words “herein,” “above” and “below” and words ofsimilar import, when used in this application, shall refer to thisapplication as a whole and not to any particular portions of thisapplication.

Terms used herein may be preceded and/or followed by a single dash, “-”,or a double dash, “=”, to indicate the bond order of the bond betweenthe named substituent and its parent moiety; a single dash indicates asingle bond and a double dash indicates a double bond. In the absence ofa single or double dash it is understood that a single bond is formedbetween the substituent and its parent moiety; further, substituents areintended to be read “left to right” unless a dash indicates otherwise.For example, C₁-C₆alkoxycarbonyloxy and —OC(O)C₁-C₆alkyl indicate thesame functionality; similarly arylalkyl and -alkylaryl indicate the samefunctionality.

The term “alkenyl” as used herein, means a straight or branched chainhydrocarbon containing from 2 to 10 carbons, unless otherwise specified,and containing at least one carbon-carbon double bond. Representativeexamples of alkenyl include, but are not limited to, ethenyl,2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl,2-heptenyl, 2-methyl-1-heptenyl, 3-decenyl, and3,7-dimethylocta-2,6-dienyl.

The term “alkyl” as used herein, means a straight or branched chainhydrocarbon containing from 1 to 10 carbon atoms, unless otherwisespecified. Representative examples of alkyl include, but are not limitedto, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, andn-decyl. When an “alkyl” group is a linking group between two othermoieties, then it may also be a straight or branched chain; examplesinclude, but are not limited to —CH₂—, —CH₂CH₂—, —CH₂CH₂CHC(CH₃)—,—CH₂CH(CH₂CH₃)CH₂—.

The term “alkynyl” as used herein, means a straight or branched chainhydrocarbon group containing from 2 to 10 carbon atoms and containing atleast one carbon-carbon triple bond. Representative examples of alkynylinclude, but are not limited, to acetylenyl, 1-propynyl, 2-propynyl,3-butynyl, 2-pentynyl, and 1-butynyl.

The term “aryl,” as used herein, means a phenyl (i.e., monocyclic aryl),a bicyclic ring system containing at least one phenyl ring or anaromatic bicyclic ring containing only carbon atoms in the aromaticbicyclic ring system or a multicyclic aryl ring system, provided thatthe bicyclic or multicyclic aryl ring system does not contain aheteroaryl ring when fully aromatic. The bicyclic aryl can be azulenyl,naphthyl, or a phenyl fused to a monocyclic cycloalkyl, a monocycliccycloalkenyl, or a monocyclic heterocyclyl. The bicyclic aryl isattached to the parent molecular moiety through any carbon atomcontained within the phenyl portion of the bicyclic system, or anycarbon atom with the napthyl or azulenyl ring. The fused monocycliccycloalkyl or monocyclic heterocyclyl portions of the bicyclic aryl areoptionally substituted with one or two oxo and/or thia groups.Representative examples of the bicyclic aryls include, but are notlimited to, azulenyl, naphthyl, dihydroinden-1-yl, dihydroinden-2-yl,dihydroinden-3-yl, dihydroinden-4-yl, 2,3-dihydroindol-4-yl,2,3-dihydroindol-5-yl, 2,3-dihydroindol-6-yl, 2,3-dihydroindol-7-yl,inden-1-yl, inden-2-yl, inden-3-yl, inden-4-yl, dihydronaphthalen-2-yl,dihydronaphthalen-3-yl, dihydronaphthalen-4-yl, dihydronaphthalen-1-yl,5,6,7,8-tetrahydronaphthalen-1-yl, 5,6,7,8-tetrahydronaphthalen-2-yl,2,3-dihydrobenzofuran-4-yl, 2,3-dihydrobenzofuran-5-yl,2,3-dihydrobenzofuran-6-yl, 2,3-dihydrobenzofuran-7-yl,benzo[d][1,3]dioxol-4-yl, benzo[d][1,3]dioxol-5-yl,2H-chromen-2-on-5-yl, 2H-chromen-2-on-6-yl, 2H-chromen-2-on-7-yl,2H-chromen-2-on-8-yl, isoindoline-1,3-dion-4-yl,isoindoline-1,3-dion-5-yl, inden-1-on-4-yl, inden-1-on-5-yl,inden-1-on-6-yl, inden-1-on-7-yl, 2,3-dihydrobenzo[b][1,4]dioxan-5-yl,2,3-dihydrobenzo[b][1,4]dioxan-6-yl,2H-benzo[b][1,4]oxazin3(4H)-on-5-yl, 2H-benzo[b][1,4]oxazin3(4H)-on-6-yl, 2H-benzo[b][1,4]oxazin3(4H)-on-7-yl,2H-benzo[b][1,4]oxazin3 (4H)-on-8-yl, benzo[d]oxazin-2(3H)-on-5-yl,benzo[d]oxazin-2(3H)-on-6-yl, benzo[d]oxazin-2(3H)-on-7-yl,benzo[d]oxazin-2(3H)-on-8-yl, quinazolin-4(3H)-on-5-yl,quinazolin-4(3H)-on-6-yl, quinazolin-4(3H)-on-7-yl,quinazolin-4(3H)-on-8-yl, quinoxalin-2(1H)-on-5-yl,quinoxalin-2(1H)-on-6-yl, quinoxalin-2(1H)-on-7-yl,quinoxalin-2(1H)-on-8-yl, benzo[d]thiazol-2(3H)-on-4-yl,benzo[d]thiazol-2(3H)-on-5-yl, benzo[d]thiazol-2(3H)-on-6-yl, and,benzo[d]thiazol-2(3H)-on-7-yl. In certain embodiments, the bicyclic arylis (i) naphthyl or (ii) a phenyl ring fused to either a 5 or 6 memberedmonocyclic cycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, or a 5or 6 membered monocyclic heterocyclyl, wherein the fused cycloalkyl,cycloalkenyl, and heterocyclyl groups are optionally substituted withone or two groups which are independently oxo or thia. Multicyclic arylgroups are a phenyl ring (base ring) fused to either (i) one ring systemselected from the group consisting of a bicyclic aryl, a bicycliccycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or(ii) two other ring systems independently selected from the groupconsisting of a phenyl, a bicyclic aryl, a monocyclic or bicycliccycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic orbicyclic heterocyclyl, provided that when the base ring is fused to abicyclic cycloalkyl, bicyclic cycloalkenyl, or bicyclic heterocyclyl,then the base ring is fused to the base ring of the a bicycliccycloalkyl, bicyclic cycloalkenyl, or bicyclic heterocyclyl. Themulticyclic aryl is attached to the parent molecular moiety through anycarbon atom contained within the base ring. In certain embodiments,multicyclic aryl groups are a phenyl ring (base ring) fused to either(i) one ring system selected from the group consisting of a bicyclicaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclicheterocyclyl; or (ii) two other ring systems independently selected fromthe group consisting of a phenyl, a monocyclic cycloalkyl, a monocycliccycloalkenyl, and a monocyclic heterocyclyl, provided that when the basering is fused to a bicyclic cycloalkyl, bicyclic cycloalkenyl, orbicyclic heterocyclyl, then the base ring is fused to the base ring ofthe a bicyclic cycloalkyl, bicyclic cycloalkenyl, or bicyclicheterocyclyl. Examples of multicyclic aryl groups include but are notlimited to anthracen-9-yl and phenanthren-9-yl.

The term “arylalkyl” and “-alkylaryl” as used herein, means an arylgroup, as defined herein, appended to the parent molecular moietythrough an alkyl group, as defined herein. Representative examples ofarylalkyl include, but are not limited to, benzyl, 2-phenylethyl,3-phenylpropyl, and 2-naphth-2-ylethyl.

The terms “cyano” and “nitrile” as used herein, mean a —CN group.

The term “cycloalkyl” as used herein, means a monocyclic, bicyclic, or amulticyclic cycloalkyl ring system. Monocyclic ring systems are cyclichydrocarbon groups containing from 3 to 8 carbon atoms, where suchgroups can be saturated or unsaturated, but not aromatic. In certainembodiments, cycloalkyl groups are fully saturated. Examples ofmonocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl,cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.Bicyclic cycloalkyl ring systems are bridged monocyclic rings or fusedbicyclic rings. Bridged monocyclic rings contain a monocyclic cycloalkylring where two non-adjacent carbon atoms of the monocyclic ring arelinked by an alkylene bridge of between one and three additional carbonatoms (i.e., a bridging group of the form —(CH₂)_(w)—, where w is 1, 2,or 3). Representative examples of bicyclic ring systems include, but arenot limited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane,bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, andbicyclo[4.2.1]nonane. Fused bicyclic cycloalkyl ring systems contain amonocyclic cycloalkyl ring fused to either a phenyl, a monocycliccycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or amonocyclic heteroaryl. The bridged or fused bicyclic cycloalkyl isattached to the parent molecular moiety through any carbon atomcontained within the monocyclic cycloalkyl ring. Cycloalkyl groups areoptionally substituted with one or two groups which are independentlyoxo or thia. In certain embodiments, the fused bicyclic cycloalkyl is a5 or 6 membered monocyclic cycloalkyl ring fused to either a phenylring, a 5 or 6 membered monocyclic cycloalkyl, a 5 or 6 memberedmonocyclic cycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a5 or 6 membered monocyclic heteroaryl, wherein the fused bicycliccycloalkyl is optionally substituted by one or two groups which areindependently oxo or thia. Multicyclic cycloalkyl ring systems are amonocyclic cycloalkyl ring (base ring) fused to either (i) one ringsystem selected from the group consisting of a bicyclic aryl, a bicyclicheteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and abicyclic heterocyclyl; or (ii) two other rings systems independentlyselected from the group consisting of a phenyl, a bicyclic aryl, amonocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl,a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclicheterocyclyl. The multicyclic cycloalkyl is attached to the parentmolecular moiety through any carbon atom contained within the base ring.In certain embodiments, multicyclic cycloalkyl ring systems are amonocyclic cycloalkyl ring (base ring) fused to either (i) one ringsystem selected from the group consisting of a bicyclic aryl, a bicyclicheteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and abicyclic heterocyclyl; or (ii) two other rings systems independentlyselected from the group consisting of a phenyl, a monocyclic heteroaryl,a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclicheterocyclyl. Examples of multicyclic cycloalkyl groups include, but arenot limited to tetradecahydrophenanthrenyl, perhydrophenothiazin-1-yl,and perhydrophenoxazin-1-yl.

“Cycloalkenyl” as used herein refers to a monocyclic, bicyclic, or amulticyclic cycloalkenyl ring system. Monocyclic ring systems are cyclichydrocarbon groups containing from 3 to 8 carbon atoms, where suchgroups are unsaturated (i.e., containing at least one annularcarbon-carbon double bond), but not aromatic. Examples of monocyclicring systems include cyclopentenyl and cyclohexenyl. Bicycliccycloalkenyl rings are bridged monocyclic rings or a fused bicyclicrings. Bridged monocyclic rings contain a monocyclic cycloalkenyl ringwhere two non-adjacent carbon atoms of the monocyclic ring are linked byan alkylene bridge of between one and three additional carbon atoms(i.e., a bridging group of the form —(CH₂)_(w)—, where w is 1, 2, or 3).Representative examples of bicyclic cycloalkenyls include, but are notlimited to, norbornenyl and bicyclo[2.2.2]oct-2-enyl. Fused bicycliccycloalkenyl ring systems contain a monocyclic cycloalkenyl ring fusedto either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl,a monocyclic heterocyclyl, or a monocyclic heteroaryl. The bridged orfused bicyclic cycloalkenyl is attached to the parent molecular moietythrough any carbon atom contained within the monocyclic cycloalkenylring. Cycloalkenyl groups are optionally substituted with one or twogroups which are independently oxo or thia. Multicyclic cycloalkenylrings contain a monocyclic cycloalkenyl ring (base ring) fused to either(i) one ring system selected from the group consisting of a bicyclicaryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicycliccycloalkenyl, and a bicyclic heterocyclyl; or (ii) two rings systemsindependently selected from the group consisting of a phenyl, a bicyclicaryl, a monocyclic or bicyclic heteroaryl, a monocyclic or bicycliccycloalkyl, a monocyclic or bicyclic cycloalkenyl, and a monocyclic orbicyclic heterocyclyl. The multicyclic cycloalkenyl is attached to theparent molecular moiety through any carbon atom contained within thebase ring. IN certain embodiments, multicyclic cycloalkenyl ringscontain a monocyclic cycloalkenyl ring (base ring) fused to either (i)one ring system selected from the group consisting of a bicyclic aryl, abicyclic heteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, anda bicyclic heterocyclyl; or (ii) two rings systems independentlyselected from the group consisting of a phenyl, a monocyclic heteroaryl,a monocyclic cycloalkyl, a monocyclic cycloalkenyl, and a monocyclicheterocyclyl.

The term “halo” or “halogen” as used herein, means —Cl, —Br, —I or —F.

The term “haloalkyl” as used herein, means at least one halogen, asdefined herein, appended to the parent molecular moiety through an alkylgroup, as defined herein. Representative examples of haloalkyl include,but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl,pentafluoroethyl, and 2-chloro-3-fluoropentyl.

The term “heteroaryl,” as used herein, means a monocyclic, bicyclic, ora multicyclic heteroaryl ring system. The monocyclic heteroaryl can be a5 or 6 membered ring. The 5 membered ring consists of two double bondsand one, two, three or four nitrogen atoms and optionally one oxygen orsulfur atom. The 6 membered ring consists of three double bonds and one,two, three or four nitrogen atoms. The 5 or 6 membered heteroaryl isconnected to the parent molecular moiety through any carbon atom or anynitrogen atom contained within the heteroaryl. Representative examplesof monocyclic heteroaryl include, but are not limited to, furyl,imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyridinyl,pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl,thiadiazolyl, thiazolyl, thienyl, triazolyl, and triazinyl. The bicyclicheteroaryl consists of a monocyclic heteroaryl fused to a phenyl, amonocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclicheterocyclyl, or a monocyclic heteroaryl. The fused cycloalkyl orheterocyclyl portion of the bicyclic heteroaryl group is optionallysubstituted with one or two groups which are independently oxo or thia.When the bicyclic heteroaryl contains a fused cycloalkyl, cycloalkenyl,or heterocyclyl ring, then the bicyclic heteroaryl group is connected tothe parent molecular moiety through any carbon or nitrogen atomcontained within the monocyclic heteroaryl portion of the bicyclic ringsystem. When the bicyclic heteroaryl is a monocyclic heteroaryl fused toa phenyl ring or a monocyclic heteroaryl, then the bicyclic heteroarylgroup is connected to the parent molecular moiety through any carbonatom or nitrogen atom within the bicyclic ring system. Representativeexamples of bicyclic heteroaryl include, but are not limited to,benzimidazolyl, benzofuranyl, benzothienyl, benzoxadiazolyl,benzoxathiadiazolyl, benzothiazolyl, cinnolinyl,5,6-dihydroquinolin-2-yl, 5,6-dihydroisoquinolin-1-yl, furopyridinyl,indazolyl, indolyl, isoquinolinyl, naphthyridinyl, quinolinyl, purinyl,5,6,7,8-tetrahydroquinolin-2-yl, 5,6,7,8-tetrahydroquinolin-3-yl,5,6,7,8-tetrahydroquinolin-4-yl, 5,6,7,8-tetrahydroisoquinolin-1-yl,thienopyridinyl, 4,5,6,7-tetrahydrobenzo[c][1,2,5]oxadiazolyl, and6,7-dihydrobenzo[c][1,2,5]oxadiazol-4(5H)-onyl. In certain embodiments,the fused bicyclic heteroaryl is a 5 or 6 membered monocyclic heteroarylring fused to either a phenyl ring, a 5 or 6 membered monocycliccycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 memberedmonocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl,wherein the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups areoptionally substituted with one or two groups which are independentlyoxo or thia. The multicyclic heteroaryl group is a monocyclic heteroarylring (base ring) fused to either (i) one ring system selected from thegroup consisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclicheterocyclyl, a bicyclic cycloalkenyl, and a bicyclic cycloalkyl; or(ii) two ring systems selected from the group consisting of a phenyl, abicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic orbicyclic heterocyclyl, a monocyclic or bicyclic cycloalkenyl, and amonocyclic or bicyclic cycloalkyl. The multicyclic heteroaryl group isconnected to the parent molecular moiety through any carbon atom ornitrogen atom contained within the base ring. In certain embodiments,multicyclic heteroaryl groups are a monocyclic heteroaryl ring (basering) fused to either (i) one ring system selected from the groupconsisting of a bicyclic aryl, a bicyclic heteroaryl, a bicyclicheterocyclyl, a bicyclic cycloalkenyl, and a bicyclic cycloalkyl; or(ii) two ring systems selected from the group consisting of a phenyl, amonocyclic heteroaryl, a monocyclic heterocyclyl, a monocycliccycloalkenyl, and a monocyclic cycloalkyl. Examples of multicyclicheteroaryls include, but are not limited to5H-[1,2,4]triazino[5,6-b]indol-5-yl,2,3,4,9-tetrahydro-1H-carbazol-9-yl, 9H-pyrido[3,4-b]indol-9-yl,9H-carbazol-9-yl, acridin-9-yl,

The term “heteroarylalkyl” and “-alkylheteroaryl” as used herein, meansa heteroaryl, as defined herein, appended to the parent molecular moietythrough an alkyl group, as defined herein. Representative examples ofheteroarylalkyl include, but are not limited to, fur-3-ylmethyl,1H-imidazol-2-ylmethyl, 1H-imidazol-4-ylmethyl, 1-(pyridin-4-yl)ethyl,pyridin-3-ylmethyl, pyridin-4-ylmethyl, pyrimidin-5-ylmethyl,2-(pyrimidin-2-yl)propyl, thien-2-ylmethyl, and thien-3-ylmethyl.

The term “heterocyclyl” as used herein, means a monocyclic, bicyclic, ormulticyclic heterocycle. The monocyclic heterocycle is a 3, 4, 5, 6 or 7membered ring containing at least one heteroatom independently selectedfrom the group consisting of O, N, and S where the ring is saturated orunsaturated, but not aromatic. The 3 or 4 membered ring contains 1heteroatom selected from the group consisting of O, N and S. The 5membered ring can contain zero or one double bond and one, two or threeheteroatoms selected from the group consisting of O, N and S. The 6 or 7membered ring contains zero, one or two double bonds and one, two orthree heteroatoms selected from the group consisting of O, N and S. Themonocyclic heterocycle is connected to the parent molecular moietythrough any carbon atom or any nitrogen atom contained within themonocyclic heterocycle. Representative examples of monocyclicheterocycle include, but are not limited to, azetidinyl, azepanyl,aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl,1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl,isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl,oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl,piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl,pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl,thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl,1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, andtrithianyl. The bicyclic heterocycle is a monocyclic heterocycle fusedto either a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl,a monocyclic heterocycle, or a monocyclic heteroaryl. The bicyclicheterocycle is connected to the parent molecular moiety through anycarbon atom or any nitrogen atom contained within the monocyclicheterocycle portion of the bicyclic ring system. Representative examplesof bicyclic heterocyclyls include, but are not limited to,2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indolin-1-yl,indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl,decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, andoctahydrobenzofuranyl. Heterocyclyl groups are optionally substitutedwith one or two groups which are independently oxo or thia. In certainembodiments, the bicyclic heterocyclyl is a 5 or 6 membered monocyclicheterocyclyl ring fused to phenyl ring, a 5 or 6 membered monocycliccycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 memberedmonocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl,wherein the bicyclic heterocyclyl is optionally substituted by one ortwo groups which are independently oxo or thia. Multicyclic heterocyclylring systems are a monocyclic heterocyclyl ring (base ring) fused toeither (i) one ring system selected from the group consisting of abicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicycliccycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ringssystems independently selected from the group consisting of a phenyl, abicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic orbicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and amonocyclic or bicyclic heterocyclyl. The multicyclic heterocyclyl isattached to the parent molecular moiety through any carbon atom ornitrogen atom contained within the base ring. In certain embodiments,multicyclic heterocyclyl ring systems are a monocyclic heterocyclyl ring(base ring) fused to either (i) one ring system selected from the groupconsisting of a bicyclic aryl, a bicyclic heteroaryl, a bicycliccycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or(ii) two other rings systems independently selected from the groupconsisting of a phenyl, a monocyclic heteroaryl, a monocycliccycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.Examples of multicyclic heterocyclyl groups include, but are not limitedto 10H-phenothiazin-10-yl, 9,10-dihydroacridin-9-yl,9,10-dihydroacridin-10-yl, 1 OH-phenoxazin-10-yl,10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl,1,2,3,4-tetrahydropyrido[4,3-g]isoquinolin-2-yl,12H-benzo[b]phenoxazin-12-yl, and dodecahydro-1H-carbazol-9-yl.

The term “nitro” as used herein, means a —NO₂ group.

The term “oxo” as used herein means a ═O group.

The term “saturated” as used herein means the referenced chemicalstructure does not contain any multiple carbon-carbon bonds. Forexample, a saturated cycloalkyl group as defined herein includescyclohexyl, cyclopropyl, and the like.

The term “thia” as used herein means a ═S group.

The term “unsaturated” as used herein means the referenced chemicalstructure contains at least one multiple carbon-carbon bond, but is notaromatic. For example, a unsaturated cycloalkyl group as defined hereinincludes cyclohexenyl, cyclopentenyl, cyclohexadienyl, and the like.

As used herein, the phrase “pharmaceutically acceptable salt” refers toboth pharmaceutically acceptable acid and base addition salts andsolvates. Such pharmaceutically acceptable salts include salts of acidssuch as hydrochloric, phosphoric, hydrobromic, sulfuric, sulfinic,formic, toluenesulfonic, methanesulfonic, nitric, benzoic, citric,tartaric, maleic, hydroiodic, alkanoic such as acetic,HOOC—(CH₂)_(n)—COOH where n is 0-4, and the like. Non-toxicpharmaceutical base addition salts include salts of bases such assodium, potassium, calcium, ammonium, and the like. Those skilled in theart will recognize a wide variety of non-toxic pharmaceuticallyacceptable addition salts.

Synthetic Methods

The two exemplary synthetic routes described below can be used togenerate derivatives that contain varying substituents at the 1- and3-positions of the pyrazolopyrimidine core (Schemes 1 and 2). The firstseries of analogs retain the naphthylmethylene substituent at the3-position and contain various alkyl groups at the 1-position. Synthesisof these analogs began with conversion of 1-naphthalene acetyl chlorideto pyrazolopyrimidine 4 in five steps (Scheme 1). (16)

To introduce various alkyl substituents at the 1-position, 4 can bederivatized with alkyl halides by direct nucleophilic displacement(5a-5e) or with alcohols using standard Mitsunobu reaction conditions(5a-5e). (17, 18) Notably, the use of support-bound triphenylphosphinefor the Mitsunobu reaction can expedite the purification of alkylatedproducts. (19) The N-Boc protecting groups can be removed frompiperidine analogues 5f-5h and the resultant free amines can beacetylated, sulfonylated, or alkylated.

The second series of derivatives can be generated with a synthetic routethat allows various substituents to be introduced at the 3-position ofthe pyrazolopyrimidine core (Scheme 2).

Iodinated pyrazolopyrimidine 6 can be generated in three steps fromcommercially available 5-amino-1H-pyrazole-4-carbonitrile. Various arylsubstituents can be introduced by coupling boronic acids and boronicesters to 6 using standard palladium-catalyzed cross-couplingconditions.

Synthetic Examples

Commercially available reagents and anhydrous solvents were used withoutfurther purification unless otherwise specified. The final purity of allcompounds was determined by analytical HPLC with an Agilent ZORBAXSB-C18 column (2.1 mm×150 mm). Analytical HPLC data were generated byinjecting 20 μL of sample solution in methanol and acetonitrile to areverse phase HPLC system run over 30 min (2-100% acetonitrile/waterwith 0.05% TFA and 2-100% methanol/water with 0.05% TFA). The productswere detected by UV at the detection frequency of 254 nm. All compoundswere determined to be >95% pure by this method. Flash chromatography wasperformed on prepacked columns of silica gel (Varian SF10-4g, Si 50) byIntelliFlash with EtOAc/hexanes or MeOH/CH₂Cl₂ as eluent. Thepurification by preparative HPLC was performed on Varian DynamaxMicrosorb 100-5 C18 column (250 mm×21.4 mm) with CH₃CN/H₂O or MeOH/H₂Oand 0.05% TFA as eluent. The mass spectra were recorded with the BrukerEsquire Liquid Chromatograph—Ion Trap Mass Spectrometer. NMR spectrawere recorded with either a Bruker 300 MHz spectrometer or Bruker 500MHz spectrometer at ambient temperature with the residual solvent peaksas internal standards. The chemical shifts are given in ppm (δ) andcoupling constants are reported in Hz. ¹H resonances are referenced toCDCl₃ (7.26 ppm), DMSO-d₆ (2.54) or CD₃OD (3.34).

Example 1 Preparation of Intermediate A

To malononitrile (0.646 g, 9.8 mmol) in THF (15 mL) and sodium hydride(0.494 g, 19.5 mmol, 95% dispersion in paraffin oil),1-naphthyleneacetyl chloride (2.0 g, 9.8 mmol) was added dropwise at5-10° C. After 3 h, the reaction mixture was warmed to room temperatureand hydrochloric acid (45 mL) was added. The mixture was then extractedwith ethyl acetate (3×75 mL) and the organic layer was dried over Na₂SO₄to yield A (1.71 g, 75%). ¹H NMR (CDCl₃, 300 MHz) δ 7.96-8.02 (m, 1H),7.80-7.91 (m, 2H), 7.49-7.59 (m, 2H), 7.41-7.47 (m, 2H), 4.12 (s, 2H),3.56 (s, 1H).

Example 2 Preparation of Intermediate B

To sodium hydrogen carbonate (6.112 g, 72.7 mmol) in 1,4-dioxane (12 mL)and water (2 mL), compound A (2.130 g, 9.1 mmol) and dimethyl sulfate(6.038 mL, 63.6 mmol) were added slowly. After stirring at 85° C. for2.5 h, water (60 mL) was added. Extraction with tert-butyl methyl ether(4×50 mL), drying of the organic layer with Na₂SO₄, and silica gelchromatography using an ethyl acetate-hexanes gradient yielded B (0.964g, 43%). ¹H NMR (CDCl₃, 300 MHz) δ 8.08-8.13 (m, 1H), 7.99-8.03 (m, 1H),7.92-7.97 (m, 1H), 7.58-7.70 (m, 2H), 7.49-7.56 (m, 1H), 7.25-7.30 (m,1H), 4.63 (s, 2H), 3.98 (s, 3H).

Example 3 Preparation of Compound 4

To compound B (0.908 g, 3.66 mmol) in ethanol (10 mL), hydrazine hydrate(222 L, 3.66 mmol, 80% hydrazine hydrate) and triethylamine (501 μL,3.66 mmol) were added. After refluxing for 3.5 h the solvent wasevaporated and water was added. The solid was collected and without anyfurther purification, was refluxed in formamide (6 mL) overnight. Thecold reaction mixture was diluted with water and the precipitate wascollected. This precipitate was redissolved in chloroform and thenpurified by silica gel chromatography using a methanol-chloroformgradient to yield 4 (456 mg, 45%). 1H NMR (DMSO-d₆, 300 MHz) δ 8.37 (s,1H), 8.05-8.14 (m, 1H), 7.89-7.97 (m, 1H), 7.78-7.86 (m, 1H), 7.38-7.57(m, 4H), 4.86 (s, 2H).

Example 4 General Procedure A

A mixture of 4 (0.073 mmol), K₂CO₃ (4.0 equiv), and the appropriatealkyl halide (1.1 equiv) were dissolved in dimethylformamide (400 μL)and acetonitrile (1 mL). The reaction was heated to 5° C. below theboiling point of the alkyl halide overnight. Preparative HPLC with amethanol/water gradient was used to yield the final compound.

Example 5 Preparation of Compound 3

3 was synthesized using isopropyl iodide as the alkyl halide in GeneralProcedure A. ¹H NMR (CDCl₃, 300 MHz) δ 8.04 (s, 1H), 7.98-8.03 (m, 1H),7.84-7.92 (m, 2H), 7.40-7.56 (m, 3H), 7.30-7.36 (m, 1H), 5.14 (sept,J=6.9, 1H), 4.76 (s, 2H), 1.62 (d, J=6.9, 6H). MS (ESI) (M+H)⁺=318.4.

Example 6 Preparation of Compound 5a

5a was synthesized using methyl iodide as the alkyl halide in GeneralProcedure A. ¹H NMR (CDCl₃, 300 MHz) δ 8.10 (s, 1H), 7.90-8.02 (m, 3H),7.46-7.61 (m, 3H), 7.36-7.42 (m, 1H), 4.79 (s, 2H), 4.14 (s, 3H).(M+H)⁺=290.4.

Example 7 Preparation of Compound 5b

5b was synthesized using propargyl bromide as the alkyl halide inGeneral Procedure A. ¹H NMR (CDCl₃, 300 MHz) δ 8.12 (s, 1H), 7.95-8.00(m, 1H), 7.87-7.92 (m, 2H), 7.46-7.55 (m, 3H), 7.36-7.40 (m, 1H), 5.24(m, 2H), 4.79 (s, 2H), 2.48 (m, 1H). MS (ESI) (M+H)⁺=314.4.

Example 8 Preparation of Compound 5c

5c was synthesized using iodoacetamide as the alkyl halide in GeneralProcedure A. ¹H NMR (CD₃OD, 500 MHz) δ 8.31 (s, 1H), 8.08-8.11 (m, 1H),7.88-7.92 (s, 1H), 7.81-7.84 (m, 1H), 7.50-7.54 (m, 2H), 7.41-7.46 (m,1H), 7.26-7.30 (m, 1H), 5.06 (s, 2H), 4.79 (s, 2H). MS (ESI)(M+H)⁺=333.4.

Example 9 Preparation of Compound 5e

5e was synthesized using benzyl bromide as the alkyl halide in GeneralProcedure A. ¹H NMR (CDCl₃, 300 MHz) δ 8.26 (s, 1H), 8.12-8.16 (m, 2H),7.88-7.94 (m, 2H), 7.74-7.80 (m, 1H), 7.61-7.65 (m, 2H), 7.40 (m, 5H),5.65 (s, 2H) 4.79 (s, 2H). MS (ESI) (M+H)⁺=366.5.

Example 10 Preparation of Compound 5f

5f was synthesized using cyclohexyl iodide as the alkyl halide inGeneral Procedure A. ¹H NMR (CD₃OD, 300 MHz) δ 8.18-8.23 (m, 1H), 8.16(s, 1H), 7.88-7.93 (m, 1H), 7.78-7.83 (m, 1H), 7.49-7.54 (m, 2H),7.36-7.43 (m, 1H), 7.16-7.21 (m, 1H), 4.79 (s, 2H), 4.18-4.24 (m, 1H),3.66-3.71 (m, 2H), 3.53-3.58 (m, 2H), 1.92-1.99 (m, 2H), 1.35-1.40 (m,2H), 0.86-0.94 (m, 2H). MS (ESI) (M+H)⁺=358.5.

Example 11 General Procedure B

A mixture of 4 (0.073 mmol), polymer-supported triphenylphosphine (2equiv of 1.52 mmol/g PL-TPP from Varian Polymer Laboratories) and theappropriate alcohol (1.0 equiv) were dissolved in dry THF (1 mL) andstirred under N2. Diethyl azodicarboxylate (1.5 equiv) was then added at0° C. After 15 min, the reaction mixture was warmed to room temperatureand allowed to run overnight. The resin was filtered off and washed withdicholoromethane, concentrated in vacuo, and purified usingreverse-phase preparative HPLC with a methanol/water gradient. Thedesired product was collected and subsequently deprotected by stirringin a mixture of trifluoroactetic acid (0.5 mL) and dicholoromethane (0.5mL) for 2 h. The reaction mixture was concentrated in vacuo to yield thefinal compound.

Example 12 Preparation of Compound 5g

5g was synthesized using N-boc piperidine-4-ol as the alcohol in GeneralProcedure B. ¹H NMR (DMSO-d₆, 300 MHz) δ 8.33 (s, 1H), 8.15-8.20 (m,1H), 7.91-7.96 (m, 1H), 7.80-7.84 (m, 1H), 7.51-7.57 (m, 2H), 7.37-7.44(m, 1H), 7.19-7.23 (m, 1H), 4.95 (m, 1H), 4.84 (s, 2H), 3.32-3.41 (m,2H), 3.05-3.20 (m, 2H), 2.11-2.26 (m, 2H), 1.95-2.05 (m, 2H). MS (ESI)(M+H)⁺=359.4.

Example 13 Preparation of Compound 5h

5h was synthesized using N-boc piperidine-4-methanol as the alcohol inGeneral Procedure B. ¹H NMR (CD₃OD, 500 MHz) δ 8.12 (s, 1H), 8.04-8.09(m, 1H), 7.93-7.97 (m, 1H), 7.81-7.85 (m, 1H), 7.48-7.52 (m, 2H),7.40-7.44 (m, 1H), 7.29-7.32 (m, 1H), 4.75 (s, 2H), 4.16 (d, J=7.0, 2H),2.83-2.90 (m, 2H), 2.17-2.24 (m, 1H), 1.70-1.76 (m, 2H), 1.36-1.47 (m,2H), 0.84-0.97 (m, 2H). MS (ESI) (M+H)⁺⁼373.5.

Example 14 Preparation of Compound 5i

5i was synthesized using N-boc piperidine-4-ethanol as the alcohol inGeneral Procedure B. ¹H NMR (CD₃OD, 300 MHz) δ 8.25 (s, 1H), 7.98-8.02(m, 1H), 7.86-7.90 (m, 1H), 7.78-7.82 (m, 1H), 7.44-7.49 (m, 2H),7.38-7.42 (m, 1H), 7.28-7.32 (m, 1H), 4.81 (s, 2H), 4.30-4.35 (m, 2H),3.57-3.62 (m, 2H), 2.50-2.61 (m, 2H), 1.90-2.01 (m, 2H), 1.75-1.85 (m,5H). MS (ESI) (M+H)⁺=387.5.

Example 15 General Procedure C

Acetic anhydride (1 equiv) and diisopropylethylamine (3 equiv) wereadded to compounds 5g-5i (0.028 mmol) and dissolved in dimethylformamide(200 μL). The reaction was allowed to stir at room temperatureovernight. The reaction mixture was diluted with methanol and water andpurified using reverse-phase preparative HPLC with a methanol/watergradient.

Example 16 Preparation of Compound 5j

5j was synthesized using 5g in General Procedure C. ¹H NMR (CDCl₃, 300MHz) δ 8.09 (s, 1H), 7.88-8.00 (m, 3H), 7.44-7.58 (m, 3H), 7.34-7.39 (m,1H), 4.95-5.05 (m, 1H), 4.81-4.90 (m, 1H), 4.76 (s, 2H), 4.04-4.13 (m,1H), 3.29-3.41 (m, 1H), 2.81-2.91 (m, 1H), 2.06-2.40 (m, 7H). MS (ESI)(M+H)⁺=401.5.

Example 17 Preparation of Compound 5k

5k was synthesized using 5h in General Procedure C. ¹H NMR (CD₃OD, 300MHz) δ 8.25 (s, 1H), 8.04-8.10 (m, 1H), 7.88-7.93 (m, 1H), 7.81-7.85 (m,1H), 7.47-7.52 (m, 2H), 7.39-7.45 (m, 1H), 7.29-7.33 (m, 1H), 4.78 (s,2H), 4.38-4.46 (m, 1H), 4.24 (d, J=6.9, 2H), 3.79-3.87 (m, 1H),2.95-3.05 (m, 1H), 2.45-2.58 (m, 1H), 2.10-2.20 (m, 1H), 2.05 (s, 3H),1.46-1.60 (m, 2H), 1.32-1.40 (m, 2H). MS (ESI) (M+H)⁺=415.5.

Example 18 Preparation of Compound 5l

5l was synthesized using 5i in General Procedure C. ¹H NMR (CD₃OD, 300MHz) δ 8.26 (s, 1H), 8.05-8.11 (m, 1H), 7.89-7.94 (m, 1H), 7.82-7.86 (m,1H), 7.48-7.54 (m, 2H), 7.41-7.46 (m, 1H), 7.30-7.34 (m, 1H), 4.82 (s,2H), 4.33-4.40 (m, 2H), 3.72-3.77 (m, 1H), 3.43-3.49 (m, 1H), 2.71-2.79(m, 1H), 2.26-2.33 (m, 1H), 2.04 (s, 3H), 1.72-1.79 (m, 2H), 1.60-1.70(m, 2H), 1.32-1.42 (m, 3H). MS (ESI) (M+H)⁺=429.5.

Example 19 General Procedure D

Acetaldehyde (10 equiv), sodium triacetoxyborohydride (3 equiv),diisopropylethylamine (0.75 equiv), and a catalytic amount of aceticacid were added to 5g-5i (0.028 mmol) and dissolved in dichloroethane(200 μL). The reaction was allowed to stir at room temperatureovernight. The reaction mixture was diluted with methanol and water andpurified using reverse-phase preparative HPLC with a methanol/watergradient.

Example 20 Preparation of Compound 5m

5m was synthesized using 5g in General Procedure D. ¹H NMR (CD₃OD, 300MHz) δ 8.22 (s, 1H), 8.08-8.14 (m, 1H), 7.87-7.92 (m, 1H), 7.80-7.85 (m,1H), 7.49-7.54 (m, 2H), 7.42-7.47 (m, 1H), 7.31-7.35 (m, 1H), 4.89-4.96(m, 1H), 4.80 (s, 2H), 4.32-4.38 (m, 2H), 3.52-3.58 (m, 2H), 2.99-3.10(m, 2H), 1.87-1.95 (m, 2H), 1.73-1.80 (m, 2H), 1.28-144 (m, 3H). MS(ESI) (M+H)⁺=387.5.

Example 21 Preparation of Compound 5n

5n was synthesized using 5h in General Procedure D. ¹H NMR (CD₃OD, 300MHz) δ 8.22 (s, 1H), 8.08-8.14 (m, 1H), 7.89-7.93 (m, 1H), 7.81-7.85 (m,1H), 7.48-7.54 (m, 2H), 7.39-7.45 (m, 1H), 7.28-7.33 (m, 1H), 4.78 (s,2H), 4.23-4.27 (m, 2H), 3.64-3.69 (m, 2H), 3.10-3.19 (m, 2H), 2.80-2.87(m, 1H), 2.17-2.24 (m, 2H), 1.78-1.84 (m, 2H), 1.57-1.66 (m, 2H),1.28-1.42 (m, 3H). MS (ESI) (M+H)⁺=401.5.

Example 22 Preparation of Compound 5o

5o was synthesized using 5i in General Procedure D. ¹H NMR (CD₃OD, 300MHz) δ 8.26 (s, 1H), 8.03-8.09 (m, 1H), 7.88-7.94 (m, 1H), 7.81-7.86 (m,1H), 7.48-7.54 (m, 2H), 7.40-7.46 (m, 1H), 7.29-7.34 (m, 1H), 4.76 (s,2H), 4.33-4.41 (m, 2H), 3.66-3.72 (m, 1H), 3.53-3.60 (m, 2H), 3.30-3.38(m, 2H), 3.02-3.10 (m, 2H), 2.39-2.51 (m, 2H), 1.86-1.97 (m, 2H),1.73-1.82 (m, 2H), 1.28-1.42 (m, 3H). MS (ESI) (M+H)⁺=415.6.

Example 23 General Procedure E

Methylsulfonyl chloride (1.0 equiv) and diisopropylethylamine (3.0equiv) were added to 5g-5i (0.028 mmol) and dissolved indimethylformamide (200 μL). The reaction was allowed to stir at roomtemperature overnight. The reaction mixture was diluted with methanoland water and purified using reverse-phase preparative HPLC with amethanol/water gradient.

Example 24 Preparation of Compound 5g

5p was synthesized using 5g in General Procedure E. ¹H NMR (CDCl₃, 300MHz) δ 8.30 (s, 1H), 8.08-8.14 (m, 1H), 7.87-7.92 (m, 1H), 7.80-7.85 (m,1H), 7.46-7.54 (m, 2H), 7.39-7.45 (m, 1H), 7.25-7.30 (m, 1H), 4.89-4.96(m, 1H), 4.80 (s, 2H), 3.64-3.73 (m, 2H), 2.94-3.05 (m, 2H), 2.76 (s,3H), 2.13-2.28 (m, 2H), 2.00-2.10 (m, 2H). MS (ESI) (M+H)⁺⁼437.6.

Example 25 Preparation of Compound 5q

5q was synthesized using 5h in General Procedure E. ¹H NMR (CD₃OD, 300MHz) δ 8.26 (s, 1H), 8.04-8.11 (m, 1H), 7.88-7.94 (m, 1H), 7.80-7.86 (m,1H), 7.48-7.54 (m, 2H), 7.39-7.45 (m, 1H), 7.29-7.35 (m, 1H), 4.78 (s,2H), 4.26 (d, J=6.9, 2H), 3.59-3.69 (m, 2H), 2.78 (s, 3H), 2.57-2.68 (m,2H), 1.98-2.06 (m, 1H), 1.55-1.63 (m, 2H), 1.34-1.40 (m, 2H). MS (ESI)(M+H)⁺=451.6.

Example 26 Preparation of Compound 5r

5r was synthesized using 5i in General Procedure E. ¹H NMR (CD₃OD, 300MHz) δ 8.26 (s, 1H), 8.03-8.09 (m, 1H), 7.88-7.94 (m, 1H), 7.81-7.86 (m,1H), 7.48-7.54 (m, 2H), 7.40-7.46 (m, 1H), 7.29-7.34 (m, 1H), 4.76 (s,2H), 4.34-4.41 (t, J=6.9, 2H), 3.50-3.59 (m, 2H), 2.76 (s, 3H),2.31-2.43 (m, 2H), 1.66-1.81 (m, 4H), 1.35-1.40 (m, 1H), 1.10-1.17 (m,2H). MS (ESI) (M+H)⁺=465.6.

Example 27 Preparation of Compound 5d

To compound B (0.107 g, 0.43 mmol) in ethanol (2 mL), t-butyl hydrazinehydrochloride (0.054 g, 0.43 mmol) and triethylamine (59 μL, 0.43 mmol)were added. After refluxing for 3.5 h, the solvent was evaporated andwater was added. The solid was collected and then refluxed in formamide(6 mL) overnight. The cold reaction mixture was diluted with water andthe precipitate was collected. This precipitate was re-dissolved inchloroform and then purified by silica gel chromatography using amethanol-chloroform gradient to yield 5d (0.025 g, 18%). ¹H NMR(DMSO-d₆, 300 MHz) δ 8.24-8.32 (m, 2H), 7.93 (m, 1H), 7.81 (m, 1H),7.52-7.58 (m, 2H), 7.39 (m, 1H), 7.13 (m, 1H), 4.81 (s, 2H), 1.66 (s,9H). MS (ESI) (M+H)⁺=332.4.

Example 28 Preparation of Intermediate C

Commercially available 5-amino-1H-pyrazole-4-carbonitrile (2.0 g, 18.5mmol) was mixed with formamide (10 mL) and heated to 180° C. overnightunder nitrogen. The solution was cooled to rt, 60 mL of water was added,and the resulting precipitate was collected by vacuum filtration toyield C (2.367 g, 95%). ¹H NMR (DMSO-d₆, 300 MHz) δ 8.13 (s, 1H), 8.06(s, 1H).

Example 29 Preparation of Intermediate D

Compound C (2.367 g, 17.5 mmol) and N-iodosuccinimide (4.810 g, 21.4mmol) were added to dimethylformamide (60 mL) and stirred at 50° C. for24 hours. Another batch of N-iodosuccinimide (0.871 g, 3.8 mmol) wasadded to the reaction mixture and was allowed to stir for an additional24 hours. The reaction mixture was cooled to room temperature and water(100 mL) was added, forming a precipitate that was collected byfiltration to yield D (4.1 g, 89%). ¹H NMR (DMSO-d₆, 300 MHz) δ 8.18 (s,1H).

Example 30 Preparation of Compound 6

A mixture of D (1.0 g, 3.8 mmol), K₂CO₃ (2.135 g, 15.4 mmol), andisopropyl iodide (421 μL, 4.2 mmol) were dissolved in dimethylformamide(7.5 mL). The reaction was heated to 80° C. and left to run overnight.An extraction with saturated sodium acetate and ethyl acetate wasperformed. The organic layer was dried with sodium sulfate andconcentrated in vacuo. The crude product was purified using silica gelchromatography with a methanol/dichloromethane gradient to yield 6 (673mg, 58%). ¹H NMR (DMSO-d₆, 300 MHz) δ 8.19 (s, 1H), 4.93-5.03 (m, 1H),1.43 (d, J=6.9, 6H).

Example 31 General Procedure F

Compound 6 (1 equiv), tetrakis(triphenylphosphine) palladium(0) (0.05equiv), and sodium carbonate (2.3 equiv) were added to the appropriateboronic acid or pinacol ester (1.1 equiv) in dimethoxyethane (1 mL) andwater (0.5 mL). The reaction was performed in a microwave at 85° C. forone hour, followed by extraction with ethyl acetate and water. Theorganic layer was collected and concentrated in vacuo to yield the crudeproduct. The crude product was then purified via flash chromatographywith an ethyl acetate/hexanes gradient.

Example 32 Preparation of Compound 7a

7a was synthesized using 4-chlorophenylboronic acid in General ProcedureF. ¹H NMR (CDCl₃, 300 MHz) δ 8.32 (s, 1H), 7.57 (m, 4H), 5.19 (sep,J=6.8, 1H), 1.61 (d, J=6.8, 6H). MS (ESI) (M+H)⁺=288.8.

Example 33 Preparation of Compound 7b

7b was synthesized using 3-chlorophenylboronic acid in General ProcedureF. ¹H NMR (CDCl₃, 300 MHz) δ 8.40 (s, 1H), 7.76 (s, 1H), 7.60 (m, 1H),7.46-7.50 (m, 2H), 5.21 (sep, J=6.7, 1H), 1.62 (d, J=6.7, 6H). MS (ESI)(M+H)⁺=288.8.

Example 34 Preparation of Compound 7c

7c was synthesized using 3, 4-dichlorophenylboronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.41 (s, 1H), 7.86 (s, 1H),7.56-7.66 (m, 2H), 5.21 (sep, J=6.7, 1H), 1.61 (d, J=6.6, 6H). MS (ESI)(M+H)⁺=323.3.

Example 35 Preparation of Compound 7d

7d was synthesized using 4-isopropylphenylboronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.38 (s, 1H), 7.64 (d, J=8.0,2H), 7.41 (d, J=8.0, 2H), 5.20 (sep, J=6.8, 1H), 3.01 (sep, J=6.8, 1H),1.61 (d, J=6.6, 6H), 1.32 (d, J=6.9, 6H). MS (ESI) (M+H)⁺⁼296.4.

Example 36 Preparation of Compound 7e

7e was synthesized using 3-isopropylphenylboronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.38 (s, 1H), 7.38-7.68 (m, 4H),5.20 (sep, J=6.8, 1H), 3.01 (sep, J=6.8, 1H), 1.62 (d, J=6.8, 6H), 1.32(d, J=6.8, 6H). MS (ESI) (M+H)⁺=296.4.

Example 37 Preparation of Compound 7f

7f was synthesized using 4-methylphenylboronic acid in General ProcedureF. ¹H NMR (CDCl₃, 300 MHz) δ 8.33 (s, 1H), 7.59 (d, J=7.7, 2H), 7.36 (d,J=7.7, 2H), 5.20 (sep, J=6.8, 1H), 2.46 (s, 3H), 1.62 (d, J=6.8, 6H). MS(ESI) (M+H)⁺⁼268.4.

Example 38 Preparation of Compound 7g

7g was synthesized using 3-methylphenylboronic acid in General ProcedureF. ¹H NMR (CDCl₃, 300 MHz) δ 8.37 (s, 1H), 7.59 (m, 3H), 7.31 (s, 1H),5.16 (sep, J=6.8, 1H), 2.43 (s, 3H), 1.61 (d, J=6.6, 6H). MS (ESI)(M+H)⁺=268.4.

Example 39 Preparation of Compound 7h

7h was synthesized using 3, 4-dimethylphenylboronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.37 (s, 1H), 7.50 (m, 2H), 7.31(s, 1H), 5.19 (sep, J=6.6, 1H), 2.37 (s, 3H), 2.35 (s, 3H), 1.61 (d,J=6.6, 6H). MS (ESI) (M+H)⁺=282.4.

Example 40 Preparation of Compound 7i

7i was synthesized using 4-fluoro-3-methylphenylboronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.38 (s, 1H), 7.45-7.58 (m, 2H),7.15 (m, 1H), 5.19 (sep, J=6.8, 1H), 2.39 (s, 3H), 1.61 (d, J=6.7, 6H).MS (ESI) (M+H)⁺=286.4.

Example 41 Preparation of Compound 7j

7j was synthesized using 4-methoxyphenylboronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.37 (s, 1H), 7.59 (m, 2H), 7.08(m, 2H), 5.19 (sept, J=6.8, 1H), 3.90 (s, 3H), 1.61 (d, J=6.8, 6H). MS(ESI) (M+H)⁺=284.4.

Example 42 Preparation of Compound 7k

7k was synthesized using 3, 4-dimethoxyphenylboronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.38 (s, 1H), 7.21-7.27 (m, 2H),7.02-7.06 (m, 1H), 5.19 (sep, J=6.9, 1H), 3.97 (m, 6H), 1.62 (d, J=6.9,6H). MS (ESI) (M+H)⁺⁼314.4.

Example 43 Preparation of Compound 7l

7l was synthesized using 3,4,5-trimethoxyphenylboronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.39 (s, 1H), 6.91 (s, 2H), 5.22(sep, J=6.9, 1H), 3.95 (s, 6H), 3.92 (s, 3H), 1.62 (d, J=6.9, 6H). MS(ESI) (M+H)⁺=344.5.

Example 44 Preparation of Compound 7m

7m was synthesized using 4-methoxy-3-methylphenylboronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.37 (s, 1H), 7.49 (m, 2H), 6.99(m, 1H), 5.19 (sep, J=6.7, 1H), 3.91 (s, 3H), 2.32 (s, 3H), 1.61 (d,J=6.7, 6H). MS (ESI) (M+H)⁺=298.4.

Example 45 Preparation of Compound 7n

7n was synthesized using 3-acetylphenylboronic acid in General ProcedureF. ¹H NMR (CDCl₃, 300 MHz) δ 8.41 (s, 1H), 8.32 (s, 1H), 8.08 (d, J=8.2,1H), 7.95 (d, J=8.2, 1H), 7.67 (m, 1H), 5.23 (sep, J=6.8, 1H), 2.70 (s,3H), 1.63 (d, J=6.6, 6H). MS (ESI) (M+H)⁺=296.4.

Example 46 Preparation of Compound 7o

7o was synthesized using naphthalene-1-boronic acid in General ProcedureF. ¹H NMR (DMSO-d₆, 300 MHz δ 8.30 (s, 1H), 8.03-8.11 (m, 2H), 7.85-7.90(m, 1H), 7.50-7.69 (m, 4H), 5.13 (septet, J=6.8, 1H), 1.53 (d, J=6.8,6H). MS (ESI) (M+H)⁺⁼304.4.

Example 47 Preparation of Compound 7p

7p was synthesized using naphthalene-2-boronic acid in General ProcedureF. ¹H NMR (CDCl₃, 300 MHz) δ 8.42 (s, 1H), 8.19 (s, 1H), 7.84-8.04 (m,4H), 7.56-7.62 (m, 2H), 5.25 (septet, J=6.7, 1H), 1.66 (d, J=6.7, 6H).MS (ESI) (M+H)⁺=304.4.

Example 48 Preparation of Compound 7q

7q was synthesized using 6-methoxynaphthalene-2-boronic acid in GeneralProcedure F. ¹H NMR (300 MHz, CDCl₃) δ 8.27 (s, 1H), 8.10 (s, 1H),7.77-7.94 (m, 2H), 7.64-7.73 (m, 2H), 7.21 (s, 1H), 5.23 (septet, J=6.9,1H), 3.98 (s, 3H), 1.65 (d, J=6.9, 6H). MS (ESI) (M+H)⁺=334.4.

Example 49 Preparation of Compound 7r

7r was synthesized using 6-ethoxynaphthalene-2-boronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.39 (s, 1H), 8.10 (s, 1H),7.76-7.92 (m, 3H), 7.19-7.23 (m, 2H), 5.23 (sep, J=6.6, 1H), 4.21 (q,J=6.9, 2H), 1.61 (d, J=6.6, 6H), 1.52 (t, J=6.9, 3H). MS (ESI)(M+H)⁺=348.4.

Example 50 Preparation of Compound 7s

7s was synthesized using 2-methoxynapthalene-3-boronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.38 (s, 1H), 8.03 (s, 1H),7.80-7.89 (m, 2H), 7.50-7.57 (m, 2H), 7.40-7.46 (m, 1H), 5.23 (sep,J=6.8, 1H), 3.98 (s, 3H), 1.66 (d, J=6.8, 6H). MS (ESI) (M+H)⁺=334.4.

Example 51 Preparation of Compound 7t

7t was synthesized using quinoline-3-boronic acid in General ProcedureF. ¹H NMR (CDCl₃, 300 MHz) δ 9.27 (s, 1H), 8.46 (m, 1H), 8.38 (s, 1H),8.17 (d, J=8.4, 1H), 7.91 (d, J=8.4, 1H), 7.77 (m, 1H), 7.62 (m, 1H),5.22 (sep, J=6.9, 1H), 1.61 (d, J=6.9, 6H). MS (ESI) (M+H)⁺⁼305.4.

Example 52 Preparation of Compound 7u

7u was synthesized using 3,4-methylenedioxyphenylboronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.37 (s, 1H), 7.14-7.21 (m, 2H),6.95-7.00 (m, 1H), 6.06 (s, 2H), 5.18 (sep, J=6.7, 1H), 1.61 (d, J=6.7,6H). MS (ESI) (M+H)⁺⁼298.4.

Example 53 Preparation of Compound 7v

7v was synthesized using 1,4-benzodioxane-6-boronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 500 MHz) δ 8.30 (s, 1H), 7.18 (s, 1H),7.06-7.14 (m, 2H), 5.20 (sep, J=6.7, 1H), 4.36 (s, 4H), 1.63 (d, J=6.7,6H). MS (ESI) (M+H)⁺⁼312.4.

Example 54 Preparation of Compound 7w

7w was synthesized using 3,4-dihydrochromen-6-ylboronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.35 (s, 1H), 7.35-7.41 (m, 2H),6.92-6.98 (m, 1H), 5.18 (sep, J=6.7, 1H), 4.27 (t, J=5.2, 2H), 2.89 (t,J=5.2, 2H), 2.03-2.12 (m, 2H), 1.61 (d, J=6.7, 6H). MS (ESI)(M+H)⁺=310.4.

Example 55 Preparation of Compound 7x

7x was synthesized using dibenzo[b,d]thiophen-4-ylboronic acid inGeneral Procedure F. ¹H NMR (CDCl₃, 500 MHz) δ 8.47 (s, 1H), 8.27 (s,1H), 7.72 (d, J=7.6, 1H), 7.66 (d, J=7.6, 1H), 7.54 (s, 1H), 7.47 (t,J=7.6, 1H), 7.39 (t, J=7.6, 1H), 5.24 (sep, J=6.8, 1H), 1.65 (d, J=6.8,6H). MS (ESI) (M+H)⁺=360.5.

Example 56 Preparation of Compound 7y

7y was synthesized using 3-Benzylphenylboronic acid pinacol ester inGeneral Procedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.35 (s, 1H), 7.56 (m,1H), 7.42-7.52 (m, 2H), 7.29-7.37 (m, 3H), 7.16-7.27 (m, 3H), 5.19 (sep,J=6.7, 1H), 4.08 (s, 2H), 1.61 (d, J=6.7, 6H). MS (ESI) (M+H)⁺=344.4.

Example 57 Preparation of Compound 7z

7z was synthesized using 3-Biphenylboronic acid in General Procedure F.¹H NMR (CDCl₃, 300 MHz) δ 8.40 (s, 1H), 7.94 (s, 1H), 7.61-7.75 (m, 5H),7.40-7.53 (m, 3H), 5.22 (sep, J=6.8, 1H), 1.64 (d, J=6.8, 6H). MS (ESI)(M+H)⁺=330.4.

Example 58 Preparation of Compound 7aa

7aa was synthesized using 3-Benzyloxyphenylboronic acid in GeneralProcedure F. ¹H NMR (300 MHz, CDCl₃) δ 8.40 (s, 1H), 8.10 (s, 1H),7.37-7.55 (m, 5H), 7.15-7.26 (m, 3H), 5.14-5.27 (m, 3H), 1.64 (d, J=6.8,6H). MS (ESI) (M+H)⁺=360.5.

Example 59 Preparation of Compound 7ab

7ab was synthesized using 3-Methylthiophenylboronic acid in GeneralProcedure F. ¹H NMR (CDCl₃, 300 MHz) δ 8.38 (s, 1H), 7.56 (s, 1H),7.36-7.50 (m, 3H), 5.21 (sep, J=6.7, 1H), 2.56 (s, 3H), 1.63 (d, J=6.7,6H). MS (ESI) (M+H)⁺=300.4.

Example 60 General Procedure G (Compounds 1 and 2)

Intermediates E:

To malononitrile (1.0 equiv) in THF (15 mL) and sodium hydride (2.0equiv, 95% dispersion in paraffin oil), the appropriate acyl chloride(1.0 equiv) was added dropwise at 5-10° C. After 3 hours, the reactionmixture was warmed to room temperature and hydrochloric acid (45 mL) wasadded. The mixture was extracted with ethyl acetate (3×75 mL) and theorganic layer was dried over Na2SO4 to yield intermediate E.

Intermediates F:

To sodium hydrogen carbonate (8.0 equiv) in 1,4-dioxane (12 mL) andwater (2 mL), compound E (1.0 equiv) and dimethyl sulfate (7.0 equiv)were added slowly. After stirring at 85° C. for 2.5 h, water (60 mL) wasadded. Extraction with tert-butyl methyl ether (4×50 mL), drying of theorganic layer with Na2SO4, and silica gel chromatography using an ethylacetate/hexanes gradient yielded intermediate F.

Compounds 1 and 2:

To compound F (1.0 equiv) in ethanol (2 mL), t-butyl hydrazinehydrochloride (1.0 equiv) and triethylamine (1.0 equiv) were added.After refluxing for 3.5 h, the solvent was evaporated and water wasadded. The solid was collected and then refluxed in formamide (6 mL)overnight. The cold reaction mixture was diluted with water and theprecipitate was collected. This precipitate was re-dissolved inchloroform and then purified by silica gel chromatography using amethanol-chloroform gradient to yield final compound.

1 was synthesized using 1-naphthoyl chloride as the acyl chloride inGeneral Procedure G. ¹H NMR (CDCl₃, 300 MHz) δ 8.25 (s, 1H), 7.97-8.10(m, 2H), 7.77-7.85 (m, 1H), 7.55-7.67 (m, 4H), 1.91 (s, 9H). MS (ESI)(M+H)⁺=318.4.

2 was synthesized using 2-naphthoyl chloride as the acyl chloride inGeneral Procedure G. ¹H NMR (DMSO-d₆, 300 MHz) δ 8.33 (s, 1H), 8.19 (s,1H), 7.98-8.10 (m, 3H), 7.78-7.83 (m, 1H), 7.57-7.62 (m, 2H), 1.79 (s,9H). MS (ESI) (M+H)⁺=318.4.

Example 61 Preparation of Intermediate G

Phthalimide (354 mg, 2.35 mmol), 3-chloropyrazin-2-yl methanol (284 mg,1.96 mmol), and resin bound triphenylphosphine (1.55 g, 2.35 mmol) wereadded to anhydrous THF. To this reaction mixture, DIAD (507 mg, 2.35mmol) was added dropwise. After 12 h the reaction mixture wasconcentrated. The crude product was purified using silica gelchromatography with a hexane/dichloromethane gradient to yieldIntermediate G (300 mg, 56% yield). ¹H-NMR (300 MHz, CDCl₃) δ 7.87-7.97(m, 2H), 8.29-8.34 (m, 2H). MS (ESI) (M+H)⁺274.2.

Example 62 Preparation of Intermediate H

Anhydrous hydrazine (0.05 mL, 1.37 mmol) was added to a solution ofIntermediate G (150 mg, 0.5 mmol) in 3.0 mL of dichloromethane at rtunder an inert atmosphere. After 2.5 h, 4.5 mL of MeOH was added and thereaction mixture was stirred at reflux for 19 h. The reaction mixturewas cooled to rt and the white precipitate that formed was filtered off.The precipitate was washed with ether (3×) and the filtrate wasconcentrated in vacuo. The resultant solid was re-dissolved in EtOAc andfiltered again. The solution was concentrated to afford 77 mg (97%yield) of pure product. ¹H-NMR (300 MHz, MeOD) δ 4.08 (s, 2H), 8.34 (d,1H, J=2.7 Hz), 8.58 (d, 1H, J=2.7 Hz). MS (ESI) (M+H)⁺143.9.

Example 63 Intermediate I

Intermediate H (26 mg, 0.19 mmol), isobutyric acid (0.02 mL, 0.2 mmol),EDCI (50 mg, 0.25 mmol), DIEA (0.1 mL, 0.5 mmol), and DMAP (5.7 mg,0.038 mmol) were dissolved in 2.0 mL of dichloromethane. The reactionmixture was stirred at rt overnight and then concentrated in vacuo. Theresultant residue was dissolved in EtOAc, washed with water (2×), NaHCO₃(1×), and brine (1×). The organic layer was dried over Na₂SO₄, andconcentrated in vacuo to afford 37 mg (93% yield) of pure IntermediateI. ¹H-NMR (300 MHz, CDCl3) δ 1.21-1.26 (m, 6H), 2.46-2.59 (m, 1H), 4.69(d, 2H, J=4.5 Hz), 8.32-8.34 (m, 1H), 8.46 (d, 1H, J=2.4 Hz).

Example 64 Intermediate J

POCl₃ (0.08 ml, 0.90 mmol) was added to Intermediate I (37 mg, 0.18mmol) dissolved in 0.78 mL of acetonitrile. A drop of DMF was added andthe reaction mixture was stirred at 55° C. for 30 minutes. The reactionmixture was then concentrated in vacuo and the resultant solid wasdissolved in 2 M ammonia in methanol followed by concentrating thissolution in vacuo. The resultant solid was dissolved in a minimal amountof water and the product was extracted into dichloromethane (4×). Theorganic layers were combined and washed with saturated NaHCO₃, (1×),concentrated in vacuo, and dried over Na₂SO₄. Concentration of thesolution afforded 33 mg (98% yield) of Intermediate J. ¹H-NMR (300 MHz,CDCl3) δ 1.46 (d, 6H, J=6.9 Hz), 3.29-3.38 (m, 1H), 7.33 (d, 1H, J=4.8Hz), 7.61 (d, 1H, J=5.1 Hz), 7.82 (s, 1H). MS (ESI) (M+H)⁺196.

Example 65 Intermediate K

Intermediate J (33 mg, 0.17 mmol) dissolved in 0.18 mL of DMF was addedto NIS (39 mg, 0.17 mmol) dissolved in 0.6 mL of DMF. The reactionmixture was heated to 60° C. for 3 h and then cooled to roomtemperature. The reaction mixture was partitioned between 1 M Na₂SO₃ anddichloromethane. The aqueous layer was then extracted withdichloromethane (3×). The organic layers were combined, dried overNa₂SO₄ and concentrated in vacuo to afford 34 mg (62% yield) of pureIntermediate K. ¹H-NMR (300 MHz, CDCl₃) δ 1.41 (d, 6H, J=6.9 Hz),3.20-3.31 (m, 1H), 7.28 (d, 1H, J=5.1 Hz), 7.65 (d, 1H, J=5.1 Hz). MS(ESI) (M+H) 322.1.

Example 66 Intermediate L

Intermediate K (33 mg, 0.10 mmol) and NH₄OH were heated to 80° C. for 6h in a microwave. The reaction mixture was concentrated in vacuo andpurified using silica gel chromatography with a hexane/EtOAc gradient toyield Intermediate L (33 mg, 66% yield). ¹H-NMR (300 MHz, CDCl₃) δ 1.4(d, 6H, J=5.4 Hz), 3.17-3.26 (m, 1H), 5.88 (br s, 2H), 7.02 (d, 1H,J=5.1 Hz), 7.23 (d, 1H, J=5.1 Hz). MS (ESI) (M+H)⁺ 303.2.

Example 67 Intermediate M

Intermediate H (104 mg, 0.76 mmol), pivalic acid (0.08 mL, 0.8 mmol),EDCI (200 mg, 1.0 mmol), DIEA (0.4 mL, 2.0 mmol), and DMAP (22 mg, 0.15mmol) were dissolved in 8.0 mL of dichloromethane. The reaction mixturewas stirred at rt overnight and then concentrated in vacuo. Theresultant residue was dissolved in EtOAc, washed with water (2×), NaHCO₃(1×), and brine (1×). The organic layer was dried over Na₂SO₄, andconcentrated in vacuo to afford 145 mg (94% yield) of pure IntermediateM. ¹H-NMR (300 MHz, CDCl₃) δ 1.35 (s, 9H), 4.59 (d, 2H, J=4.8 Hz),8.31-8.34 (m, 1H), 8.42 (d, 1H, J=2.4 Hz).

Example 68 Intermediate N

POCl₃ (0.12 ml, 1.35 mmol) was added to Intermediate M (54 mg, 0.27mmol) dissolved in 1.2 mL of acetonitrile. A drop of DMF was added andthe reaction mixture was stirred at 55° C. for 30 minutes. The reactionmixture was then concentrated in vacuo and the resultant solid wasdissolved in 2 M ammonia in methanol followed by concentrating thissolution in vacuo. The resultant solid was dissolved in a minimal amountof water and the product was extracted into dichloromethane (4×). Theorganic layers were combined and washed with saturated NaHCO₃ (1×),concentrated in vacuo, and dried over Na₂SO₄. Concentration of thesolution afforded 45 mg (95% yield) of Intermediate N. ¹H-NMR (300 MHz,CDCl₃) δ 1.35 (s, 9H), 7.38 (m, 1H), 7.66 (m, 1H), 7.87 (s, 1H). MS(ESI) (M+H)⁺210.1.

Example 69 Intermediate O

Intermediate N (45 mg, 0.25 mmol) dissolved in 0.23 mL of DMF was addedto NIS (51 mg, 0.22 mmol) dissolved in 0.8 mL of DMF. The reactionmixture was heated to 60° C. for 3 h and then cooled to roomtemperature. The reaction mixture was partitioned between 1 M Na₂SO₃ anddichloromethane. The aqueous layer was then extracted withdichloromethane (3×). The organic layers were combined, dried overNa₂SO₄ and concentrated in vacuo to afford 49 mg (70% yield) of pureIntermediate O. ¹H-NMR (300 MHz, CDCl₃) δ 1.31 (s, 9H), 7.23 (d, 1H,J=5.1 Hz), 7.67 (d, 1H, J=5.1 Hz). MS (ESI) (M+H)⁺ 336.4.

Example 70 Intermediate P

Intermediate O (76 mg, 0.22 mmol) and NH₄OH were heated to 80° C. for 6h in a microwave. The reaction mixture was concentrated in vacuo andpurified using silica gel chromatography with a hexane/EtOAc gradient toyield Intermediate P (76 mg, 66% yield). ¹H-NMR (300 MHz, CDCl₃) δ 1.32(s, 9H), 5.28 (br s, 2H), 7.06 (d, 1H, J=5.1 Hz), 7.21 (d, 1H, J=5.1Hz). MS (ESI) (M+H)⁺ 317.1.

Example 71 General Procedure H

Intermediates L or O (0.03 mmol), tetrakis(triphenylphosphine)palladium(0) (0.05 equiv), and sodium carbonate (2.3 equiv) were addedto the appropriate boronic acid or pinacol ester (1.1 equiv) indimethoxyethane (1 mL) and water (0.5 mL). The reaction was performed ina microwave at 85° C. for one hour, followed by extraction with ethylacetate and water. The organic layer was collected and concentrated invacuo to yield the crude product. The crude product was then purifiedvia flash chromatography with an ethyl acetate/hexanes gradient.

Example 72 Preparation of Compounds 8-12 and 69-72

Compounds 8-12 and 69-72 (Table B) were generated with General ProcedureH using the appropriate substituted boronic acid as is familiar to thoseskilled in the art.

Example 73 Preparation of Compounds 81-149

Compounds 81-149 were prepared according to the general procedures usingthe appropriate substituted boronic acid as is familiar to those skilledin the art.

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Example 74 Preparation of Compound 150

3-(6-ethoxynaphthalen-2-yl)-1-((1-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine(150)

This compound was synthesized as shown in the scheme below.

BIOLOGICAL EXAMPLES Biological Example 1 Enzymology of TgCDPK1

Two types of enzyme assays were developed to follow TgCDPK1 activity, aradiometric scintillation proximity assay measured the labeledγ-phosphate of ATP added to a biotinylated peptide substrate and an ATPconsumption assay where ATP consumption was monitored by luciferase andlight production (KinaseGlo®, Promega Corp., Madison, Wis.). Both assaysgave similar results for calcium dependence, K_(m) of substrates (lessthan 2-fold differences in Km values; see Table 6 FIGS. 6, 7, 8, and 9),and inhibitor concentrations for 50% enzyme inhibition (IC₅₀s; see,Tables 6 and 7).

TABLE 6 K_(m) and IC₅₀ values for TgCDPK1 enzyme Inhibitor IC₅₀ values(μM)* Enzyme Mean Substrate NA-PP2 Assay concn K_(m) NM-PP1 NA-PP1 (CmpdType (nM) ATP Peptide (Cmpd 3) (Cmpd 1) 2) SPA 2 10 ± 2 12 ± 2 0.0250.484 0.006 KinaseGlo 2 12.38 20.48 0.02 0.816 0.005 *ATP value of 12.4μM and Peptide 10 μM Note: Enzyme concentrations are 10-fold less thanshown in Table 7, substrate concentrations are different than Table 7,and the incubation time is 90 min instead of 30 min as shown in Table 7.

TABLE 7 Inhibitor IC₅₀ values at different TgCDPK1 enzyme concentrationsEnzyme incubation IC₅₀ values (μM)# concn time NM-PP1 NA-PP1 NA-PP2 (nM)(min) (Cmpd 3) (Cmpd 1) (Cmpd 2) 20 30 0.066 0.989 0.018 2 90 0.0390.978 0.008 #TgCDPK1 data at ATP value of 10 μM and Peptide 40 μM, withcoupled KinaseGlo assay. Note, a longer incubation time was necessaryfor the lower enzyme concentration to get reliable measurements for theIC₅₀ calculation.

As expected, activity of TgCDPK1 on the peptide substrates requiredaddition of exogenous calcium (FIG. 1). Indeed, calcium titrationrevealed that a concentration of 4.7 μM gave 50% of TgCDPK1 kinaseactivity. The K_(m) of ATP and peptide substrates were determined to be10 and 14 μM, respectively.

Biological Example 2 Structure of TgCDPK1

We have determined X-ray crystal structures of Ca²⁺-free TgCDPK1 in theapo form and in complex with two potent inhibitors (Table 1 and FIG. 2).

TABLE 1 Data collection and refinement statistics. SAD Apo NA-PP2 NM-PP1(SeMet) (native) (SeMet) (native) Data collection Space group P2₁ P2₁P2₁ P2₁ Cell dimensions a, b, c(Å) 48.2, 72.0, 67.5 48.1, 71.9, 67.348.1, 72.5, 67.1 47.2, 72.9, 65.7 α, β, γ (°) 90.0, 102.9, 90.0 90.0,103.0, 90.0 90.0, 103.8, 90.0 90.0, 98.8, 90.0 Resolution (Å) 50-2.30(2.38-2.30)* 50-2.04 (2.12-2.04) 50-1.98 (2.06-1.98) 50-1.99 (2.06-1.99)Unique reflections 20,099 (1,986) 28,324 (2,799) 30,0803 (2,996) 30,350(2,975) R_(merge) 0.11 (0.95) 0.05 (0.67) 0.09 (0.70) 0.06 (0.74) I/σI13.1 2.4 17.0 (2.4) 8.4 (2.1) 14.2 (1.9) Completeness (%) 100 (100) 99.9(100) 98.1 (96.6) 99.8 (98.1) Redundancy 7.7 (7.7) 4.2 (4.2) 3.7 (3.6)4.2 (4.1) Wilson B factor (Å²) 43.7 34.8 26.6 29.7 Refinement Resolution(Å) 36.89-2.04 29.72-1.98 35.72-1.99 No. reflections 26,871 29,22428,797 No. reflections test set 1,431 1,556 1,536 R_(work)/R_(free)0.213/0.258 0.193/0.245 0.192/0.228 No. atoms Protein 3,674 3,776 3,743Nonprotein 79 194 167 B-factors (Å²) Protein (Mean B_(iso) + B_(TLS))60.2 42.5 48.7 Nonprotein (Mean B_(iso)) 27.7 27.1 24.0 R.m.s.deviations Bond lengths (Å) 0.006 0.007 0.006 Bond angles (°) 0.8660.978 0.878 PDB code 3i79 3i7c 3i7b *Values in parentheses are forhighest-resolution shell.

The structure of the catalytic domain is typical of serine/threoninetype protein kinases. The two Ca²⁺-binding EF-hand lobes and connectingextended helical stem of the calmodulin-like regulatory domain lie alongone face of the kinase domain, adjacent to the active site (FIG. 2a ).The kinase and regulatory domains are connected by an interveninghelical junction domain characteristic of this class of kinases¹⁸. Theconformation represented by these structures is likely an inactive formof the enzyme since the calmodulin-like domain occludes the surfacerequired for recognition of target proteins and peptides. Importantly,the ATP binding site remains accessible to small molecule substrates andinhibitors. The group of Raymond Hui at the Toronto Structural GenomicsConsortium has shown that when TgCDPK1 is activated by calcium, theregulatory domain undergoes major structural rearrangement and isrepositioned to lie against the opposite surface of the kinase domain(Wernimont et al. submitted for publication, PDB accession number 3hx4).This dramatic structural change allows access of the protein substratesto the active site.

Biological Example 3 Bumped Kinase Inhibitors (BKIs) and TgCDPK1

Most known kinase inhibitors bind in the ATP-binding pocket of theactive site^(19,20). These inhibitors exploit many of the samehydrophobic contacts as the purine ring of ATP and make at least oneconserved hydrogen bond to the hinge region. Potent inhibitors alsooccupy at least one hydrophobic pocket adjacent to the ATP-binding site.These additional hydrophobic interactions increase both binding affinityand target selectivity of the inhibitor because there is substantialheterogeneity among different kinases in these regions. Examination ofthe TgCDPK1 sequence in the vicinity of the ATP-binding pocket (FIG. 2b) shows that it contains a glycine residue at a position that has beentermed the gatekeeper residue because it constrains access to theATP-binding site²¹⁻²³. The glycine at this position in TgCDPK1 (Gly128)is expected to create a much larger pocket off the ATP-binding site thanis typically seen in protein kinases and comparison of the TgCDPK1structure with other kinases shows that this is indeed the case. Thisdifference in the active site architectures can be exploited for designof selective inhibitors against TgCDPK1.

Shokat and colleagues have shown that mutation of bulky gatekeeperresidues to glycine renders mutant kinases uniquely susceptible toinhibition by a class of kinase inhibitors called “bumped kinaseinhibitors” (BKIs). BKIs are analogs of4-amino-1-tert-butyl-3-phenylpyrazolo[3,4-d]pyrimidine (Table 2) thatare derivatized at C3 with bulky aromatic groups²⁴.

TABLE 2 Inhibition of wt TgCDPK1 and TgCDPK1 (Met gatekeeper) by bumpedkinase inhibitors.* Inhibitor NA-PP1 NA-PP2 NM-PP1 Wild type TgCDPK1 0.989  0.018  0.066 (IC50 in μM) G128M TgCDPK1 173    21.44  36.18 (IC50 in μM) Chemical structure

*The IC₅₀ values were calculated by non-linear regression using Prism(GraphPad Software). The assay was performed three times and the meanIC50 is shown. The assay was performed using coupling with KinaseGlo at20 nM TgCDPK1, 10 μM ATP, 40 μM peptide, and an incubation time of 30min.The large sidechain of the gatekeeper residue in most kinases preventsaccess of the C3 bulky aromatic substituent to the more hydrophobicpocket at the back of the catalytic cleft rendering them insensitive toBKI inhibition^(21,22). Large gatekeeper amino acids like methionine orphenylalanine severely restrict access by the BKIs, while small residuessuch as the glycine present in TgCDPK1 are mostpermissive^(22,23,25,26). Studies with genetically engineered mice thatexpress mutant kinases with small gatekeeper residues (glycine oralanine), have demonstrated that BKIs preferentially target the mutantkinases²⁷⁻²⁹. The well-documented lack of BKIs inhibition of mammaliankinases suggests that this class of compounds may be very selective forTgCDPK1 during T. gondii infection²⁷⁻²⁹.

Based on structural and sequence analysis of the differences inATP-binding sites of typical mammalian kinases and TgCDPK1 with a smallgatekeeper (FIGS. 2a and 2b ), we synthesized three BKIs anddemonstrated that two of three were low double digit nanomolarinhibitors of TgCDPK1 (Table 2). To show that the glycine gatekeeper isthe primary determinant of BKI inhibition, we created a glycine tomethionine gatekeeper mutant of TgCDPK1 (G128M). Indeed the wild typeenzyme was inhibited significantly better by individual BKIs compared tothe G128M mutant enzyme (Table 2). For NA-PP1, there was a 175-folddifference, for NA-PP2, a 1200-fold difference and for NM-PP1, a550-fold difference between IC₅₀ values for wild type versus the G128Mmutant (Table 2).

The crystal structure of TgCDPK1 in complex with two potent BKIs weredetermined (NA-PP2, FIG. 2c ; NM-PP1, FIG. 2d ) and, as predicted, theseinhibitors bind in the ATP-binding site with the C3 bulky aromaticsubstituent occupying the pocket adjacent to the glycine gatekeeper.Superposition of an orthologous CDPK containing a methionine gatekeeperonto our BKI complexes showed impairment of the favorable binding modeobserved for the BKIs in TgCDPK1 (FIG. 2e ). The bulky gatekeepersidechain clashes with the C3 bulky aromatic substituent providing astructural basis for the insensitivity of typical kinases towards BKIinhibition, as verified experimentally with the G128M mutant of TgCDPK1(Table 2). These results strongly implicate TgCDPK1 with its uniqueglycine gatekeeper as a drug target for selective treatment oftoxoplasmosis.

Biological Example 4 TgCDPK1 Appears in the Cytoplasm and Nucleus of T.gondii Cells

The mode of action of TgCDPK1 is unknown but its localization in thecell might provide clues to its cellular function. To this end, TgCDPK1was fused with green fluorescent protein (GFP) to facilitate itslocalization within live intracellular T. gondii. TgCDPK1-GFP was foundin both the cytosol and the nucleus (FIG. 3). Similar findings wereobtained when the GFP tag was replaced with a C-terminal hemagglutinin(HA) tag. Thus CDPK1 may have targets in both cytosol and nucleus.

Biological Example 5 Effects of BKIs on Toxoplasma gondii Cell Entry andGrowth

We tested three BKIs for effects on host cell invasion and parasitegrowth since earlier work on TgCDPK1¹⁴ suggested a key role for thisenzyme in T. gondii invasion. As shown in FIG. 4, BKIs profoundlyreduced T. gondii proliferation if added simultaneously with cellularinfection (FIG. 4a ), and modestly reduced proliferation when added 4 hrafter the start of invasion (FIG. 4b ). Thus, inhibition of TgCDPK1activity by BKIs has a stronger effect on invasion than on intracellulargrowth. This was confirmed by microscopic examination of invasion (seeFIGS. 8(a)-8(c) and 9(a)-9(c)) and is consistent with previousobservations on the role of TgCDPK1^(12,14).

T. gondii expressing CDPK1-GFP, CDPK1(G128M)-GFP, or GFP were mixed withthe indicated concentration of NA-PP2 and added to the fibroblastmonolayer for 15 min at 4° C. to allow binding (Kafsack et al). Thesamples were then raised to 37° C. for 4 min to allow invasion. Sampleswere fixed, washed, and stained with antibody to T. gondii surfaceantigens (SAG, Argene) to identify extracellular parasites. FIG. 8(a) isa representative image from the untreated CDPK-GFP dataset.Intracellular parasites are marked by asterisks. FIG. 8(b) shows thechange in the number of intracellular parasites upon drug treatment. Thenumber of intracellular parasites was determined by subtracting thenumber of extracellular parasites (SAG+) by the number of totalparasites (GFP+) and expressed as a ratio to the number of host cellsobserved (as measured by DAPIstained nuclei). Each sample was normalizedto the untreated control of the same line. FIG. 8(c) shows the data usedto derive the graph in panel b. Note that cells expressing thegatekeeper mutant CDPK1(G128M) are less sensitive to the drug. The drugadditionally appears to affect adhesion.

T. gondii overexpressing CDPK1-GFP, CDPK1(G128M)-GFP, or GFP were mixedwith wild type cells (105 of each) in medium with or without 1 μMNA-PP2. Parasites were allowed to infect fibroblasts on cover slips andgrown overnight. Samples were stained with DAPI to identify host andparasite nuclei and vacuoles with 2 or more T. gondii cells (only fullyinvaded parasites can replicate) were counted. Each vacuole was assessedfor GFP expression by the parasites. FIG. 9(a) is a representative setof images of CDPK1(G128M)-GFP mixed with wild type, in the absence ofdrug. Vacuoles containing multiple GFP-expressing parasites are markedwith * and wild type vacuoles are marked with 4. FIG. 9(b) shows thenumber of vacuoles with wild type (GFP−) or transfected parasites(GFP+), per host cell nucleus, in the absence and presence of drug forthe three cell mixtures. FIG. 9(c) shows Data used to derive the graphs.Note that the parasite line overexpressing CDPK1(G128M)-GFP wasresistant to the effects of 1 μM NA-PP2, as compared to the co-culturedwild type control.

We expect that longer exposure to BKIs would increase the impact of thedrug on T. gondii growth, as egressed parasites are prevented frominvading new cells. Each BKI was tested for its effects on humanfibroblast replication; the EC₅₀s were found to be approximately1000-fold higher (not shown) than those seen in the T. gondii “invasion”assay.

Biological Example 6 T. gondii Cell Expressing the G128M GatekeeperMutant Demonstrate that BKIs Act Through TgCDPK1 In Vivo

We transfected the T. gondii cell line with expression plasmids encodingan HA tag fused to the C-terminus of either wild type TgCDPK1 or theG128M gatekeeper mutant. Immunoblot analysis showed that the wild typeand G128M mutant TgCDPK1s were expressed to similar levels (FIG. 5a ).Compared to the parental cell line, parasites expressing the G128Mmutant were relatively resistant to BKIs NA-PP2 (FIG. 5b ) and NM-PP1(data not shown) added prior to invasion. In contrast, parasitesoverexpressing the wild type TgCDPK1 protein showed only a small shiftin resistance. Although NA-PP2 was somewhat less effective againstwild-type parasites in this experiment, the results were identical inthe relative potency of the inhibitors, in that NA-PP2 was more potentthan NM-PP1 in blocking T. gondii invasion, following the potencyobserved against TgCDPK1 enzyme. Furthermore, in both a microscopicassay of invasion (see FIGS. 8(a)-8(c))³⁰ and in a mixed cellularinfection using wild type and TgCDPK1(G128M) (see FIGS. 9(a)-9(c)),parasites expressing the gatekeeper mutant were markedly resistant toNA-PP2 while cells expressing either wild type TgCDPK1 or GFP controlswere not. These findings demonstrate that BKIs are working primarilythrough the TgCDPK1 target to prevent T. gondii mammalian cell entry.

We have shown that TgCDPK1 is a promising drug target for the therapy oftoxoplasmosis. Its kinase activity is uniquely sensitive to inhibitionby BKIs, and BKI treatment in turn blocks entry of the parasite intomammalian cells. Blocking cell entry is important because T. gondii isan obligate intracellular parasite and cannot replicate withoutinvasion. Moreover, these experimental findings address concerns aboutquantitative differences between enzyme inhibition and cellular effectsthereby validating the utility of TgCDPK1 structures in complex withBKIs to drive drug development for toxoplasmosis therapy. The concept ofusing BKIs for the therapy of toxoplasmosis is bolstered by the factthat BKIs have been used in mouse studies with no demonstration oftoxicity or troublesome effects to the animals²⁷⁻²⁹. Thus, BKIs havepromise; as a selective drug for toxoplasmosis therapy because they arenon-toxic to mammals but prevent cell entry and thus the replication ofT. gondii.

Since the structure was determined for the inactive, Ca²⁺-free form ofTgCDPK, there may be some concern that this form would not be optimal toguide the design of small molecule inhibitors with improved potency andselectivity. It is apparent from structural and biochemical studiespresented here, however, that small molecule inhibitors can still accessthe ATP-binding site of the Ca²⁺-free conformation of TgCDPK1.Importantly, the structure of the active site near the ATP-bindingpocket, particularly in the vicinity of the gatekeeper residue, is notsignificantly altered between the structures shown here and that of theCa²⁺-bound enzyme in complex with AMP-PNP (Wernimont et al. submittedfor publication, PDB 3hx4). Thus, structure-guided optimization of smallmolecule inhibitors that target this region is possible using theinactive, Ca²⁺-free form of TgCDPK1.

TgCDPK1 was localized in the cytoplasm, but also found in the nucleus.Some plant CDPKs are also partially localized to the nucleus. In thoseCDPKs, nuclear localization is mediated by a signal in the junctiondomain¹⁸, but the T. gondii protein is not homologous in this region andno nuclear localization signal is predicted by standard programs. Sincethe size of the protein is above the threshold for free diffusionthrough the nuclear pore, we propose that the protein could bear anon-canonical nuclear localization sequence or piggyback into thenucleus on another protein. As noted earlier, Raymond Hui's group hasdemonstrated a Ca²⁺-dependent structural rearrangement that repositionsthe regulatory domain to the opposite side of the catalytic domain(Wernimont et al. submitted for publication, PDB accession number 3hx4).This observation of Ca²⁺-induced structural rearrangement raises theintriguing, but speculative, possibility that Ca²⁺ could modulatelocalization by revealing or occluding the relevant region of thekinase. In any case, these results raise the possibility that TgCDPK1phosphorylates specific nuclear proteins in addition to its presumablycytosolic targets involved in gliding motility.

Drug-resistant mutations of the TgCDPK1 gatekeeper to a bulky residuecould eventually emerge under selective pressure of BKI therapy. Onestrategy to suppress the emergence of resistance is theco-administration of two drugs targeting different proteins. As TgCDPK1is not the target of any existing drug, the development of ananti-TgCDPK1 compound could provide a partner drug for co-administrationwith another drug. Most transmission of T. gondii is not fromperson-to-person (although this can occur in pregnancy, transplantation,or transfusion), but rather through zoonotic cycles where drug pressureis not exerted¹. This suggests that if drug resistance emerges it willlargely be confined to the individual, posing little threat to theutility of the drug in other infected persons.

As other apicomplexan pathogens employ CDPK enzymes with a smallgatekeeper residue, this work may have broader applicability. Forinstance, the TgCDPK1 ortholog in Cryptosporidium parvum also has aglycine residue at the gatekeeper position (FIG. 2b ), suggesting thatBKIs targeting T. gondii could also be effective for the therapy ofcryptosporidiosis, another potentially life-threatening infection withpoor therapeutics. Other parasite CDPKs have small gatekeeper residues,such as threonine. Indeed, an extended search of all reported humankinase ATP binding motifs found none with a glycine or alaninegatekeeper residue, and about ˜20% with threonine²¹. Thus, thesefindings may have implications in the rational design ofanti-apicomplexan CDPK agents devoid of toxic side effects to the hostcells.

Biological Example 7 Expression, Purification and Assay of RecombinantMutant and Wild Type TgCDPK1

TgCDPK1 (GI: 12484153, ToxoDB ID 162.m00001) or its G128M mutant, clonedinto pAVA0421, was expressed in BL21*(DE3) E. coli (Invitrogen Corp.Carlsbad, USA) for 72 hours at 20° C. in a LEX Bioreactor (HarbingerBiotechnology & Engineering Corp. Ontario Canada). Cells collected from2 liters of culture were lysed in 200 ml of 30 mM imidazole, 500 mMNaCl, 25 mM HEPES, 5% glycerol, 0.025% azide, 1 mM TCEP, 10 mM MgCl₂, 1mM AEBSF, pH 7.0 and incubated with Benzonase® (Novagen, USA) for 30 minat room temperature before centrifugation at 14,000 rpm for 75 min and4° C. Clarified supernatant was loaded onto 5 ml Ni²⁺ HisTrap FF resincolumns pre-equilibrated with 30 mM imidazole, 500 mM NaCl, 25 mM HEPES,5% glycerol, 0.025% azide, 1 mM TCEP. Recombinant histidine-taggedprotein was eluted in 220 mM imidazole pH 7.0. Fractions weresubsequently separated on AKTA Prime size exclusion columns (HiLoad™26/60 Superdex 75™ prep grade) pre-equilibrated 500 mM NaCl, 25 mMHEPES, 5% glycerol, 0.025% azide, 2 mM DTT, pH 7.0. The N terminalhexahistidine tag was removed by 3C protease cleavage at a 1:50 mg ratioand dialyzed overnight at 4° C. in 200 mM NaCl, 20 mM HEPES, 5%glycerol, 1 mM TCEP, pH 7.0 with final separation on Ni²⁺ Sepharose™ 6Fast Flow resin. Fractions were analyzed for purity bySDS-polyacrylamide gel electrophoresis and found to be >99% pure.

Two types of kinase assays were employed, an ATP consumption assay and ascintillation proximity assay. ATP consumption kinase assays wereperformed using a nonradioactive KinaseGlo® luciferase assay (PromegaCorp., Madison, Wis., USA). Kinase phosphorylation reactions wereperformed in a buffered medium containing 20 mM HEPES pH 7.5 (KOH), 0.1%BSA, 10 mM MgCl₂, 1 mM EGTA (pH 7.2), plus or minus 2 mM CaCl₂ ¹⁴. Thephosphorylation reaction mixture of 40 μM peptide substrate (Syntide-2,peptide sequence:Pro-Leu-Ala-Arg-Thr-Leu-Ser-Val-Ala-Gly-Leu-Pro-Gly-Lys-Lys (SEQ ID NO:12)) (GenScript, Piscataway, USA), 19.48 nM of 14 TgCDPK1, 90 to 0.0005μM serial dilutions of inhibitor in a total volume of 25 μl, wasinitiated by the addition of 10 μM ATP. The reaction was terminatedafter 30 minutes incubation at 30° C. by addition of excess EGTA (5 mMfinal concentration). Internal positive and negative controls wereincluded in each assay run. No activity was detected when the peptidesubstrate was left out of the reaction mixture. Unused ATP was measuredin luminescence based readout as counts per seconds on the Chameleon425-104 multi-label plate scintillation counter (Hidex, Oy, TurkuFinland). A second enzyme assay, based on the scintillation proximityassay, directly measured the attachment of γ-phosphate to peptidesubstrate. The scintillation proximity assay was used for confirmationof enzymatic characteristics like K_(m) for ATP and substrate and forconfirmation of IC50s of the inhibitors. Phosphorylation of biotinatedpeptide substrate, (Bio-Syntide-2, American Peptide Company, Inc.Sunnyvale, Calif.) was determined by serial titrations in scintillationproximity assays using streptavidin coated Beads and [γ-³³P] ATP(PerkinElmer, Boston, USA) as previously described³⁰.

Biological Example 8 Site-Directed Mutagenesis

Site-directed mutagenesis of the predicted gate keeper glycine residueto methionine was executed with Stratagene kit. PCR amplification ofpAVA0421-TgCDPK1 plasmid DNA with page purified primers TgDPKmetS (GGCTAC TTC TAC CTC GTC ATG GAA GTG TAC ACG GGA GGC GAG TTG)(SEQ ID NO: 3)and TgDPKmetAS (CAA CTC GCC TCC CGT GTA CAC TTC CAT GAC GAG GTA GAA GTAGCC)(SEQ ID NO: 4) was carried out at 95° C. (30 seconds), 95° C. (30seconds), 55° C. (1 minute), 68° C. (6:00 minutes) followed by 68° C.(6:00 minutes) repeated in 16 cycles. Dpn I digested PCR products wastransformed into XL10-Gold ultracompetent E. coli cell (Stratagene) withselection on LB agar supplemented with 100 g/ml ampicillin. Mutantplasmids was recovered with alkaline denaturation method using theQIAGEN plasmid purification kits and verified by nucleotide sequenceanalysis before transformation into protein expression E. coli strainBL21*(DE3) (Invitrogen Corp. Carlsbad, USA). Induction of expression andpurification of mutant TgCDPK1(G128M) in E. coli BL21*(DE3) (InvitrogenCorp. Carlsbad, USA) was previously described³⁰.

Biological Example 9 Protein Crystallization

Purified TgCDPK1 protein was screened for crystallization leads using aPhoenix crystallization robot (Art Robbins Instruments, Sunnyvale,Calif.) and the JCSG+ Suite sparse matrix crystallization screen(Qiagen, Valencia, Calif.)³¹. A few leads were further optimized byfiner grid screening around the initial hit using sitting-drop vapordiffusion at room temperature. Diffraction-quality crystals of nativeand selenomethionyl-derivatized (SeMet) protein (˜18-26 mg/ml) weregrown from mother liquor composed of 0.18-0.25 M (di- or tri-) ammoniumcitrate (pH 6.5-7.0), 22-26% polyethylene glycol (PEG) 3350, and 5 mMdithiothreitol. For inhibitor cocrystals, the crystallization drop wasadditionally supplemented with a final concentration of 4 mM inhibitordissolved in dimethylsulfoxide. Thin pointed rods or platesapproximately 50-150 μm long typically grew within one week. Crystalswere mounted in cryoloops (Hampton Research, Aliso Viejo, Calif.) anddirectly frozen in liquid nitrogen or were first transferred to a freshdrop of mother liquor containing 10-20% ethylene glycol for, typically,less than 20 seconds prior to freezing. For inhibitor co-crystals, 2-4mM inhibitor was maintained in the cryoprotection solution.

Biological Example 10 Structure Determination

Crystals of TgCDPK1 were screened at the Stanford Synchrotron ResearchLightsource (SSRL) on beamline 9-2 using the SSRL automated mounting(SAM) system³². All data were collected at 100 K on a MarMosaic-325 CCDdetector using the Blu-Ice software package³³. Single-wavelengthanomalous dispersion (SAD) data were collected from a single crystal ofSeMet protein³⁴ at a wavelength of 0.9792 Å, the Se peak wavelengthdetermined from a fluorescence scan of the crystal. All other datasetswere also collected at or near the Se peak wavelength; apo (native) at0.9792 Å, NA-PP2 complex (SeMet) at 0.9791 Å, and NM-PP1 complex(native) at 0.9795 Å. All data were processed using HKL2000³⁵. Datacollection statistics are presented in Table 1.

Initially, we were able to solve the structure of the kinase domain bymolecular replacement (MR) using MOLREP³⁶ or Phaser³⁷ with the structureof the kinase domain of the orthologous protein from Cryptosporidiumparvum (PDB ID 3dfa) as the search model. Although representing nearlytwo-thirds of the total protein content of the asymmetric unit, theresulting MR phases were not good enough to allow tracing of the novelcalmodulin-like regulatory domain; despite significant effort at manualimprovement of the MR solution and attempts to additionally placevarious calmodulin-like domains of very low sequence identity by MR. Wethus turned to using SeMet protein in order to obtain experimental phaseinformation to solve the complete structure. Using a SeMet SAD dataset,SOLVE³⁸ located 12 of 14 expected selenium atoms and produced initialphases to approximately 2.7 Å. The resulting phases were input intoRESOLVE³⁸ using the RESOLVE_BUILD script(http://www.solve.lanl.gov/Resolve/html_resolve_manual/resolve_build.txt)for density modification and automated model building. The model wassignificantly improved by bootstrapping the results back into theRESOLVE_BUILD script two more times for rebuilding. Iterated manualmodel building and restrained refinement continued using Coot39 andREFMAC5⁴⁰. This model was transferred into the other datasets by MRusing Phaser followed by a few rounds of model building and improvementin Coot and refinement with REFMAC5. The improved models were thensubmitted to ARP/wARP⁴¹ for rebuilding using and continued cycles ofbuilding and restrained refinement ensued. Based on the initial,unbiased difference density present in the active sites of the cocrystalstructures, it was immediately clear that the inhibitors had bound theenzyme. Ideal coordinates and refinement restraints for use in REFMAC5and Coot for these ligands were created with the PRODRG server⁴². In thefinal cycles of refinement, perturbational displacement of the proteinwas described by a multi-group translation/libration/screw (TLS) model,with group boundaries suggested by the TLSMD server^(43,44), that wererefined prior to restrained refinement. Model quality was monitored andvalidated using Coot and MolProbity⁴⁵. All final models display goodRamachandran statistics. For the final apo model, 97.6% of the residuesare in favored regions of the Ramachandran plot with no outliers. TheNA-PP2 complex model has 98.9% of residues in favored regions with nooutliers and the NM-PP1 complex has 98.5% of residues in favored regionsand Gly420 is an outlier. The CCP4 suite of programs^(46,47) was usedfor all steps from data preparation through refinement. Model refinementstatistics are presented in Table 1. Structural figures were created andrendered using PyMOL (Delano Scientific, Palo Alto, Calif.).

Atomic coordinates and structure factors have been deposited in theProtein Data Bank⁴⁸. (http://www.pdb.org) with accession numbers 3i79(apo), 3i7c (NA-PP2 complex), and 3i7b (NM-PP1 complex).

Biological Example 11 T. gondii Growth/Invasion Assay and Expression ofTgCDPK1-HA and TgCDPK1 (G128M)

The TgCDPK1 coding region or its G128M mutant were amplified from the E.coli expression plasmids using primers to insert 3 adenine nucleotidesbefore the start codon and mutate the second codon from glycine toalanine, allowing for enhanced expression in the parasite⁴⁹. Theproducts were inserted into the BglII/AvrII site of T. gondiitransfection vector pCAT-GFP⁵⁰ or pHx-ACP-YFP⁵¹ to yield in framefusions with GFP or YFP, under the control of the tubA or Gra1 promotersrespectively. Plasmids encoding HA-tagged versions of the molecules werecreated by replacing the YFP sequence with one that specifies fourC-terminal HA epitopes. Linearized constructs were electroporated intoRH strain T. gondii according to standard methods⁵² and transfectedparasites were used to infect human foreskin fibroblasts growing oncover slips. In some cases a T. gondii strain expressing β-galactosidaseunder control of the TubA promoter, as well as GFP under control of theGRA1 promoter, was employed (kind gift of Gustavo Arrizabalaga)⁵³.

Immunofluorescence analysis was performed as described⁵² using rabbitanti-GFP (Invitrogen Corp. Carlsbad, USA) followed by goat anti-rabbitIgG coupled to Texas red (Southern Biotechnology). DNA was stained withDAPI. Samples were viewed on a Nikon Eclipse E600 using a Plan Apo 60X1.4 na objective.

T. gondii cells expressing β-galactosidase were grown in the presence ofserial concentrations of BKIs. All assays were performed in triplicateand the mean and standard deviation are presented unless otherwisenoted. In the growth assays, 10³ parasites were allowed to infect aconfluent layer of fibroblasts in each well of a 96 well plate. After 4hr, drug was added to the indicated final concentration. To assesseffects on invasion, compounds were added prior to the addition of theparasites. The cultures were incubated for 20 hours at 37° C. andβ-galactosidase was assayed as earlier described using chlorophenol red3-galactopyranose (CPRG, Sigma) as substrate⁵⁴. Plates were developed at37° C. for 4 hr (growth), 8 hr (invasion), or 10 hr (CDPK expression)and absorbance was measured at 595 nm on a Versamax microplate reader.

Cytotoxicity was analyzed by adding medium containing up to 50 μM ofeach compound to a confluent layer of fibroblasts; plates were incubatedat 37° C. for 4 days. Alamar blue (Alamar Biosciences, Sacramento,Calif.) was added to each well and developed over night.

Biological Example 12 CpCDPK1

The sensitivity of CpCDPK1 to compounds 1-3 was determined with an invitro activity assay (FIG. 12A). Both TgCDPK1 and CpCDPK1 show similarlevels of inhibition by these compounds, with analog 2 as the mostpotent inhibitor. Next, the phenotypic response of C. parvum cells tocompounds 1-3 was tested by measuring the amount of parasites presentafter 18 hours of infection of human intestinal cells (HCT-8) with C.parvum sporozoites in the presence of varying concentrations of theseinhibitors. These assays were performed under two different sets ofconditions: one in which the sporozoites were exposed to each inhibitorat the time of infection (FIG. 12B, left graph) and a second assay inwhich the sporozoites were exposed to each inhibitor one hour afterinfection (FIG. 12B, right graph). For each assay, the HCT-8 cells werelysed after 24 hours of culture and the amount of C. parvum rRNA wasquantified using reverse transcriptase real time PCR. The amount of C.parvum rRNA was then related to the number of parasites using standardcurves developed separately. Importantly, reduced levels of C. parvumsporozoites are observed with increasing concentrations of compounds 1-3under both sets of conditions, with analogue 2 demonstrating the mostpotent effect on parasite proliferation similar to analogue 2's superiorpotency against CpCDPK1. The potencies of the pyrazolopyrimidineinhibitors were dramatically increased when they were added at the timeof sporozoite infection rather than one hour after C. parvum had beenincubated with HCT-8 host cells. This suggests that these inhibitorsaffect an early stage of C. parvum host cell invasion, which is similarto their effects on T. gondii invasion. In all cases, the relativeability of each inhibitor to block C. parvum proliferation correlateswith its in vitro activity against CpCDPK1.

Based on the ability of pyrazolopyrimidine inhibitors to block T. gondiiand C. parvum host cell invasion, compounds were further optimized basedon this scaffold as potent and selective dual inhibitors of TgCDPK1 andCpCDPK1. Two synthetic routes were used to generate derivatives thatcontain varying substituents at the 1- and 3-positions of thepyrazolopyrimidine core as described in the preceding syntheticexamples.

Inhibition of TgCDPK1 and CpCDPK1 was determined using a luminescentkinase assay which measures ATP depletion in the presence of the Syntide2 peptide substrate (KinaseGlo). (U.S. Provisional Patent ApplicationNo. 61/299,286, and reference 9) Similar to TgCDPK1, exogenous calciumwas necessary for CpCDPK1 to possess maximum catalytic activity (datanot shown). Notably, both kinases were tested at the same ATPconcentration which allows direct comparison of inhibitor potencies dueto these enzymes possessing similar K_(m)s for this cofactor. (20)

Encouraged by the similar potency of inhibitor 3 against TgCDPK1(IC₅₀=150±20 nM) and CpCDPK1 (IC₅₀=130±40 nM), pyrazolopyrimidineanalogues that contain a naphthylmethylene group at the 3-position andvarious alkyl substituents at the 1-position were tested for theirability to inhibit both kinases (Table 3).

TABLE 3 In vitro activities of 3, 5a-5r against TgCDPK1 and CpCDPK1.Values shown are the average of three assays ± (SEM).

Com- pound Num- CpCDPK1 TgCDPK1 R₁ = ber IC₅₀ (nM) IC₅₀ (nM)

3 150 (20) 103 (40)

5a 410 (90) 460 (240)

5b 130 (30) 990 (180)

5c 2000 (400) 1900 (100)

5d 140 (50) 31 (10)

5e >3000 1800 (700)

5f 2400 (300) 1000 (300)

5g 56 (16) 59 (18)

5h 9.0 (0.5) 15 (5)

5i 34 (7) 52 (17)

5j 100 (10) 91 (26)

5k 360 (80) 210 (20)

5l 180 (60) 100 (10)

5m 50 (7) 36 (3)

5n 160 (30) 270 (30)

5o 100 (10) 93 (4)

5p 75 (18) 54 (9)

5q 700 (240) 560 (10)

5r 200 (80) 120 (30)

Derivatives containing smaller alkyl groups (5a-5c) were found to havereduced potencies for both enzymes relative to 3. Furthermore, asignificant increase in potency was not observed for inhibitors thatcontain bulkier substituents at the 1-position (5d-5f). However, thereduced potencies of compounds with larger substituents is not due to asize restriction in the ATP-binding pocket because derivatives thatcontain a basic piperidine group (derivatives 5g-5i) were found to besignificantly more potent inhibitors of TgCDPK1 and CpCDPK1. Notably,compound 5h, which contains a methylene linkage between thepyrazolopyrimidine core and piperidine ring, has an 8-fold and 16-foldlower IC₅₀ for CpCDPK1 and TgCDPK1 than parent compound 3, respectively.Derivatization of 5g-5i with an acetyl (5j-5l), ethyl (5m-5o), orsulfonylmethyl (5p-5r) group reduces the enhanced potency that thepiperidine substituent confers, with only modified versions ofpiperidine 5g showing similar inhibition as the parent compound.Notably, most compounds from this series are near equipotent inhibitorsof both TgCDPK1 and CpCDPK1, demonstrating the overall similarity inthis region of the ATP-binding pockets of both kinases.

To further probe the hydrophobic pocket adjacent to the gatekeeperresidue (Hydrophobic Pocket II) a series of analogs that contain anisopropyl group at the 1-position and various aryl substituents at the3-position were tested for their ability to inhibit both kinases (Table4, compounds 7a-7ab). While many of these inhibitors were not expectedto be as selective for CpCDPK1 and TgCDPK1 over mammalian kinases asnaphthylmethylene derivatives 3 and 5a-5r, we felt that this series ofcompounds would provide insight into which substituents can beaccommodated in this region. As shown in Table 4, a large number of arylsubstituents are accommodated by both kinases. Almost all of thecompounds from this series have an IC₅₀ less than 1 M, with severalinhibitors demonstrating very high potency against both enzymes.Inhibitors that contain aryl rings that are mono-substituted withsmaller substituents at the meta (7b, 7e, 7g, and 7ab) or para positions(7a, 7d, 7f, and 7j) are accommodated in the ATP-binding sites of bothkinases but are more potent against TgCDPK1 than CpCDPK1. However,neither kinase is inhibited by compounds that contain aryl rings thatare meta-substituted with larger substituents at the 3-position (7y, 7z,and 7aa). Analogues that contain meta- and para-substituted aryl rings(7c, 7i, 7k, and 7m) are potent against both enzymes and show reducedselectivity for TgCDPK1. Consistent with this observation,pyrazolopyrimidine derivatives that contain a 2-naphthyl (7p-7r) orquinoline group (7t) are potent inhibitors of both enzymes. However,bicyclic substituents that are not planar show reduced potency (7u-7w).In general, most inhibitors from this series are 2- to 12-fold selectivefor TgCDPK1 over CpCDPK1. However, several compounds (for example,compounds 7l, 7q and 7t) are equipotent or slightly selective forCpCDPK1. This fact demonstrates that while the ATP-binding pockets ofboth kinases are very similar they are not identical.

TABLE 4 In vitro activities of 7a-5ab against TgCDPK1 and CpCDPK1.Values shown are the average of three assays ± (SEM).

Com- pound Num- CpCDPK1 TgCDPK1 R₁ = ber IC₅₀ (nM) IC₅₀ (nM)

7a 220 (70) 18 (4)

7b 120 (40) 20 (6)

7c 13 (3) 4.0 (1.0)

7d 210 (60) 48 (14)

7e 59 (18) 14 (5)

7f 87 (25) 13 (4)

7g 114 (33) 19 (9)

7h 22 (6) 9.0 (3.0)

7i 53 (9) 9.0 (2.0)

7j 410 (130) 40 (18)

7k 170 (20) 37 (11)

7l 93 (7) 420 (70)

7m 17 (5) 5.0 (1.0)

7n 2100 (200) 1500 (400)

7o 620 (130) 330 (80)

7p 10 (3) 5.0 (1.0)

7q 5.0 (1.0) 6.0 (1.0)

7r 12 (4) 5.0 (1.0)

7s 600 (100) 900 (210)

7t 20 (4) 24 (6)

7u 310 (20) 45 (14)

7v 990 (70) 180 (20)

7w 110 (10) 17 (6)

7x >3000 >3000

7y 1400 (200) 320 (90)

7z >3000 2200 (200)

7aa 1900 (400) 1100 (200)

7ab 120 (30) 56 (17)

Key to the use of pyrazolopyrimidine-based kinase inhibitors asanti-parasitic agents is the potential to selectively inhibit TgCDPK1and CpCDPK1 over mammalian kinases. To determine the potency of thesecompounds against mammalian kinases, a subset of inhibitors were testedagainst the tyrosine kinases SRC and ABL. We felt that these two kinaseswould be a suitable counter-screen because they both contain the mostpermissive gatekeeper residue found in mammalian kinases (threonine) andthe pyrazolopyrimidine core was originally developed as a kinaseinhibitor scaffold against SRC-family kinases. (21)

As shown in Table 5, most of the pyrazolopyrimidines tested have limitedactivity against SRC and ABL. Consistent with earlier studies,previously-characterized “bumped” inhibitors 1 and 3 do not potentlyinhibit SRC or ABL kinase. Despite the increased activity of analogue 2against TgCDPK1 and CpCDPK1, this compound only weakly inhibits SRC andABL (IC₅₀>1 μM).

TABLE 5 Activities of various inhibitors against human kinases and humancell lines. Values shown are the average of three assays ± (SEM).Compound IC₅₀ (μM) GI₅₀ (μM) Number Abl Src CRL8155 HL60 HCC2998 SF5391 >20 1.3 (0.2) >10 >10 >10 >10 2 6.0 (1.5) 1.9 (0.2) >10 >10 >10 >10 37.9 (2.0) 8.8 (1.8) >10 >10 >10 >10 5d 7.7 (3.8) 2.2(0.5) >10 >10 >10 >10 5h >20 >20 >10 >10 >10 >10 5m 0.36 (.05)  0.21(.06)  — — — — 5n >20 >20 >10 >10 >10 >10 5o >20 4.2 (2.1) — — — — 5p0.090 (.019)  0.36 (.07)  >10 >10 >10 >10 7c 0.07 (.02)  0.035(.008)  >10 >10 >10 >10 7e 0.20 (.02)  0.22 (.02)  — — — — 7p 0.075(.024)  0.065 (.009)  >10 >10 ~10 >10 7q 0.82 (.04)  0.77(0.10) >10 >10 >10 >10 7r 1.7 (0.5) 0.20 (.03)  — — — — 7t 0.86 (.06) 2.7 (0.6) >10 >10 >10 >10

As expected, pyrazolopyrimidine inhibitors that contain smaller arylgroups at the 3-position (7c and 7e) are less selective for bothparasite kinases due to their increased potency against SRC and ABL.However, analogues that contain a substituted naphthyl (7q and 7r) orquinoline (7t) substituent at the 3-position are much more selective duethe increased size of these functional groups. Presumably, the largerthreonine gatekeeper residues of SRC and ABL restrict access of theseinhibitors to Hydrophobic Pocket II. While the substituent at the3-position is the major contributor to inhibitor selectivity,substitution at the 1-position affects the potency of these compoundsagainst TgCDPK1 and CpCDPK1 relative to SRC and ABL. For example,piperidine-containing analogues 5m and 5p are less selective for TgCDPK1and CpCDPK1 than parent compound 1. However, several analogues restorethe high selectivity of this class of compounds, with secondarypiperidine 5h demonstrating over 1000-fold selectivity for the CDPKs. Totest the overall toxicity of our pyrazolopyrimidines inhibitors, theywere assayed for their ability to inhibit the growth of four human celllines (Table 5): glioma derived (SF539), human lymphocyte (CRL8155),human promyelocytic leukemia (HL-60) and colorectal carcinoma (HC 2998).For all four cell lines tested, the inhibitors showed little or nogrowth inhibition at the highest concentration tested (10 μM).

We have previously reported crystal structures of inhibitors 2 and 3bound to TgCDPK1. (U.S. Provisional Patent Application No. 61/299,286,and reference 9) Similar to the structures of other pyrazolopyrimidinesbound to tyrosine kinases, these inhibitors occupy the ATP-binding cleftof TgCDPK1. The pyrazolopyrimidine scaffold superimposes with the purinering of ATP, with the exocyclic amine and nitrogen at the 5-positionforming hydrogen-bonding interactions with the hinge region. Inaddition, the glycine gatekeeper residue of TgCDPK1 allows bulkysubstituents at the 3-position unobstructed access to Hydrophobic PocketII. Replacing glycine with a larger amino acid at this position clearlycreates a steric clash with the 2-naphthyl and 1-naphthylmethylenegroups of inhibitors 2 and 3, respectively. (8) Based on the similarsensitivities of TgCDPK1 and CpCDPK1 to pyrazolopyrimidines we predictedthat inhibitors of this class would bind in a similar orientation inboth enzymes. The structure of compound 3 bound to CpCDPK1 shows thatthis is indeed the case (FIG. 13a ). Superposition of the active sitesof TgCDPK1 and CpCDPK1 bound to 3 shows that the inhibitor has anidentical mode of binding in both kinases. Furthermore, all of theresidues surrounding the inhibitor are nearly superimposable, which isremarkable because TgCDPK1 is in the calcium-free inactive conformationwhile CpCDPK1 is in the calcium-bound active conformation (FIG. 15).

It is therefore clear that the inhibitors are able to bind theATP-binding site of CDPK1 whether it is active or not.

While structures of both enzymes bound to 2 and 3 provided directinsight into how large substituents can be accommodated at the3-position, we were interested in further exploring how substitution atthe 1-position affects the potencies of these inhibitors. Structures ofinhibitor 5h bound to TgCDPK1 and CpCDPK1 show how increased potency canbe obtained. While the pyrazolopyrimidine cores and 3-positionsubstituents of inhibitors 3 and 5h make identical contacts with bothenzymes, the piperidine ring of 5h forms a salt bridge with a glutamateresidue that lines the ATP-binding cleft. Importantly, this interactionis present in the structures of 5h bound to both TgCDPK1 and CpCDPK1.Presumably, alkylation, acetylation or sulfonylation of the piperidinering disrupts this interaction and accounts for the lower potency ofcompounds 5j-5r. Furthermore, the reduced potency of 5h against SRC andABL demonstrates that these kinases cannot form a similar interaction.

In conclusion, we have demonstrated that selective pyrazolopyrimidinekinase inhibitors are indeed potent inhibitors of CpCDPK1 and that theyare able to inhibit an early stage of C. parvum cell invasion. Thisclass of compounds have a similar effect on the ability of T. gondii toinvade human fibroblast cells. (U.S. Provisional Patent Application No.61/299,286, and reference 9) Based on these results, a diverse panel ofpyrazolopyrimidine analogues was generated and their activities againstCpCDPK1 and TgCDPK1 were determined. By exploiting a unique sequence andstructural variation in the ATP-binding clefts of TgCDPK1 and TgCDPK1potent dual inhibitors of these enzymes were obtained. Importantly, manyof these inhibitors show minimal inhibition of the tyrosine kinases SRCand ABL and are not toxic to human cell lines. Additionally, otherderivatives of pyrazolopyrimidine may be useful and such variations onthe compounds discussed herein are contemplated. For example, thepotency and selectivity of these compounds and optimization theirPK/ADME/Tox properties. Furthermore, optimized inhibitors may be able tobe used to inhibit C. parvum host cell invasion.

Biological Example 13 TgCDPK1 and CpCDPK1 IC₅₀ Data

The following compounds were tested for inhibition of TgCDPK1 andCpCDPK1 (Table 8) according to the preceding methods:

TABLE 8 TgCDPK1 and CpCDPK1 IC₅₀ data IC₅₀ (μM) Cmpd # TgCDPK1 CpCDPK1 1 1.619 >3.0  2 0.015 0.059  3 0.128 0.147  5a 0.458 0.413  5b 0.0860.13  5c 1.923 2.038  5d 0.031 0.144  5e 1.848 >3.0   5f 0.946 2.416  5g0.059 0.056  5h 0.015 0.009   5i 0.052 0.034   5j 0.091 0.103  5k 0.2080.36   5l 0.104 0.179   5m 0.036 0.05  5n 0.269 0.163  5o 0.093 0.103 5p 0.054 0.075  5q 0.563 0.691   5r 0.117 0.194  7a 0.018 0.216  7b0.02 0.115  7c 0.004 0.013  7d 0.048 0.213  7e 0.014 0.059   7f 0.0130.087  7g 0.019 0.114  7h 0.009 0.022   7i 0.009 0.053   7j 0.04 0.41 7k 0.037 0.173   7l 0.42 0.093   7m 0.005 0.017  7n 1.483 2.068  7o0.326 0.62  7p 0.005 0.01  7q 0.006 0.005   7r 0.005 0.012   7s 0.8630.591   7t 0.024 0.02  7u 0.045 0.312  7v 0.181 0.984   7w 0.017 0.11 7x >3.0 >3.0  7y 0.322 1.402  7z 2.229 >3.0   7aa 1.058 1.894   7ab0.056 0.121  8 0.0048 0.0078  9 0.04 0.032 10 0.078 0.0336 11 0.01030.0573 12 0.0032 0.0532 13 0.553 0.894 14 0.627 0.953 15 0.149 0.265 160.9609 >3.0 17 0.98 1.554 18 >3.0 >3.0 19 0.0638 0.3705 20 0.0036 0.00321 >3.0 >3.0 22 1.142 >3.0 23 >3.0 >3.0 24 0.0119 0.0704 25 0.00380.0073 26 0.0091 0.0162 27 0.0569 0.6231 28 0.0049 0.0023 29 0.00380.0035 30 0.1144 0.0837 31 0.1168 0.0936 32 0.0038 0.0051 33 0.00260.0046 34 0.0023 0.0021 35 0.0037 0.0044 36 0.003 0.0032 37 0.00130.0008 38 0.0007 0.0006 39 0.0007 0.0004 40 0.0008 0.0497 41 0.00060.0041 42 0.0009 0.054 43 0.0054 0.244 44 0.0037 0.0067 45 0.015 0.09246 0.0106 0.1017 47 0.0055 0.2717 48 0.1366 1.5858 49 0.0186 0.2102 500.0522 1.0316 51 0.0159 0.2624 52 0.0043 0.3728 53 0.012 0.2723 540.0159 0.1471 55 0.0163 0.0307 56 0.004 0.1052 57 0.01 0.0134 58 0.13570.5877 59 0.0997 0.8319 60 0.0666 0.9289 61 0.0684 0.9269 62 0.04090.6918 63 0.0462 0.4168 64 0.1429 0.9285 65 0.1706 1.1576 66 0.17772.0183 67 0.1963 0.8222 68 >3 >3 69 0.0046 0.0107 70 0.0042 0.0022 710.0049 0.0053 72 0.0249 0.0123

Biological Example 14 Tyrosine Kinase IC₅₀ Data

The following compounds were tested for inhibition of human tyrosinekinases SrcKD, Src3D, AblKD, Abl3D, HckKD (Table 9) according to thepreceding methods:

TABLE 9 Tyrosine Kinase IC₅₀ data IC₅₀ (μM) Cmpd # SrcKD Src3D AblKDAbl3D HckKD  1 1.3 >20.0  2 1.9 6  3 8.8 7.9 1.382  5d 2.2 7.7 5h >20.0 >20.0 >10.0   5m 0.21 0.36  5n >20.0 >20.0  5o 4.2 >20.0  5p0.36 0.09  7c 0.035 0.07  7e 0.22 0.2  7p 0.065 0.075  7q 0.77 0.5760.82 0.375   7r 0.2 0.549 1.7 0.218   7t 0.2 1.7  9 0.686 2.01 11 0.4750.628 2.36 0.541 12 0.298 0.307 0.838 0.244 20 0.294 0.238 24 1.45 0.35325 5.02 0.969 26 1.242 0.304 28 >20.0 >20.0 >10.0 29 >20.0 >20.0 >10.030 >20.0 >20.0 32 >10.0 >10.0 >10.0 33 >10.0 >10.0 >10.0 34 3.063 >10.01.613 35 >10.0 >10.0 >10.0 36 >10.0 >10.0 >10.0 37 1.759 >10.0 38 0.3750.497 39 1.551 >10.0 40 0.038 0.043 0.316 41 0.199 0.675 42 0.784 1.6843 0.263 1.153 44 >10.0 7.34 45 >10.0 7.326 48 0.8832 1.701 49 0.11180.0333 50 0.1966 1.073 51 0.4003 1.798 52 0.1067 1.267 53 0.1911 1.23 540.1254 0.194 55 0.897 0.6492 57 >10 >10 68 >10 >10 69 3.9 >10 70 2.384.79 71 6.09 >10 72 >10 6.35 83 >10 4.159 84 1.287 0.0862 85 0.09480.403 90 0.094 91 0.01 92 >10 ~10 94 >10 0.554 99 >10 >10 118  >10 >10120  >10 >10 123  >10 >10 124  3.78 7.845 132  0.1773 2.943 134  0.57961.636 135  2.01 4.861 137  2.21 8.040 138  5.02 139  0.5210 1.589 140 0.1286 0.6356 142  2.10 143  4.13 144  >10 5.982 145  >10 >10 146 0.0366 0.172 148  5.58 149  8.68 150  >10 >3

Biological Example 15 Expression, Purification and P. falciparum EnzymeActivity Assays

Recombinant PfCDPK1 and PfCDPK4 proteins were expressed in E. coli(Rosetta Oxford strain) and lysed in 25 mM HEPES (pH 7.0), 500 mM NaCl,5% glycerol, 0.5% CHAPS, 30 mM imidazole, 1 mM TCEP, 250 μg/ml AEBSF,0.025% azide, and 2.5 U/mL benzonase nuclease (Novagen). Solublerecombinant enzymes were purified as earlier described by Ojo et al.(23), which is incorporated by reference herein in its entirety. Aluminescence assay that measures the depletion of ATP in the presence ofthe peptide substrate, Syntide 2 (PLARTLSVAGLPGKK) (24,9), was used todetermine the catalytic activity of these enzymes. Assays were performedwith 10 μM ATP, 40 μM Syntide-2, and 6.6 nM or 22 nM of PfCDPK1 andPfCDPK4, respectively, in 20 mM HEPES (pH 7.5), 0.1% BSA (w/v), 10 mMMgCl₂, 1 mM EGTA, with or without 2 mM CaCl₂. The BKI compound librarypreviously described by Murphy et al. (24), which is incorporated byreference herein in its entirety, was tested at serial concentrationsbetween 3 μM and 1 nM.

Biological Example 16 PfCDPK4 Binding Mode Modeling

The kinase domain of PfCDPK4 was modeled by the I-TASSER server (25,26)using structures of TgCDPK1 available in the Protein Data Bank as atemplate. Subsequent docking of Compound 29 into the active site of thismodel was carried out using QXP/FLO (27) (version +0602). Compound 29was initially placed in the active site of PfCDPK4 by SSM superposition(28) of an in-house TgCDPK1:Compound 29 co-crystal structure (PDBcitation pending). The binding pocket was defined as all residues within10 Å of the roughly-placed inhibitor. Protein atoms were fixed with theexception of sidechain atoms that project into the binding pocket. Thetwo H-bonds between the pyrazolopyrimidine inhibitor scaffold and thehinge region of the kinase, which are conserved in mostATP/ATP-homolog:kinase complexes, were restrained. The interactionbetween the nitrogen of the R2 piperidine and the sidechain of Glu154was also restrained because we see this interaction in >15 co-crystalstructures of TgCDPK1/CpCDPK1 with BKIs containing the methylpiperidineR2 substituent (24). Docking of Compound 29 was subsequently carried outusing 1100 cycles of Metropolis Monte Carlo conformational searchingfollowed by energy minimization. The 25 lowest energy binding modes werevisually inspected and favorable conformations were selected.

Biological Example 17 P. berghei Maintenance and Genetic Modification

The P. berghei ANKA wild type clone 2.34 and the transgenic linesderived from it were maintained in Theiler's Original (TO) or SwissWebster outbred mice and infections monitored on Giemsa-stained bloodfilms. The cdpk4⁻ mutant clone and its complementation with P. bergheicdpk4 were described previously by Billker et al., which is incorporatedby reference herein in its entirety. A transfection vector forcomplementing the cdpk4⁻ mutant with Pfcdpk4 was generated by replacingthe P. berghei sequence in plasmid p150 with a Pfcdpk4 sequenceamplified by PCR from P. falciparum 3D7 gametocyte cDNA as an NheI-ApaIrestriction fragment, placing it in frame with a carboxy terminal 2x mycepitope tag, a 3′UTR and terminator derived from the P. berghei dhfr tsgene. Following verification of the Pfcdpk4 sequence, the plasmid waslinearized in a unique HpaI site within the cdpk4 5′ intergenic regionand transfected into P. berghei cdpk4⁻ using established protocols (29).Homologous insertion of the complementation vector into the upstreamintergenic region of the cdpk4 mutant placed Pfcdpk4 under the controlof the endogenous P. berghei promoter. Transgenic clones were genotypedby diagnostic PCR followed by Southern blot analysis.

Biological Example 18 Phenotypic Analysis of P. berghei GametocytesExpressing PfCDPK4-2x-Myc and Sensitivity to Compound 29

Expression of CDPK4-2x-myc proteins was verified by western blotanalysis of mixed asexual parasite and gametocytes purified fromperipheral blood of infected mice by ammonium chloride lysis. Parasiteswere suspended and lysed in SDS loading buffer containing 0.1 M DTT.Protein blots were probed with anti-myc mouse monoclonal antibody 9E10(Sigma) at 1:2000 dilution. To assess the effect of complementation,exflagellation was quantified 3 to 4 days post infection by adding 4 μlof blood from a superficial tail vein to 150 μl exflagellation medium(RPMI1640 containing 25 mM HEPES (Sigma), 10% FCS, 100 μM xanthurenicacid, pH 7.5). Between 15 and 18 minutes after activation the number ofexflagellating microgametocytes was counted in a haemocytometer and thered blood cell (RBC) count determined. The percentage of RBCs containingmicrogametocytes was assessed on Giemsa-stained blood smears and thenumber of exflagellations per 100 microgametocytes was then calculated.Ookinetes formation was analysed in-vitro largely as described (30), byculturing gametocyte-infected mouse blood in exflagellation medium for24 h at 19° C. Conversion from macrogametocytes to ookinetes wasassessed by staining live cultures with Cy3-conjugated monoclonalantibody 13.1 against the macrogamete/ookinete surface marker P28. Theconversion rate was determined as the number of banana shaped ookinetesas a percentage of the total number of P28 expressing cells. For theexperiments monitoring exflagellation after compound administration,mice bearing P. berghei gametocytes expressing PfCDPK4-2x-myc weretreated with 50 mg/kg i.p. of BKI-2 or NA-PP2 or vehicle (90% saline 7%EtOH 3% DMSO), bled at the time points noted in FIG. 17c ,exflagellation events determined as above, and compound concentration inthe blood determined by quantitative liquid chromatography/massspectrometry.

Biological Example 19 P. berghei Transmission Experiments

Mice were injected i.p. with drug or vehicle 3 days after an infectionhad been initiated with ˜10⁶ P. berghei ANKA parasites constitutivelyexpressing GFP (31). After 30 minutes treated mice were anaesthetized byi.p. injection of a mixture of ketamine (Ketalar®, 80 mg/kg) andxylazine (Rompun®, 8 mg/kg) and exposed to 25 female A. stephensimosquitoes. After 5 days midguts were dissected, imaged using afluorescence microscope and oocysts quantified.

Biological Example 20 P. falciparum Transmission Experiments

P. falciparum strain NF54 parasites were cultured in RPMI 1640supplemented with 50 μM hypoxanthine and 10% A+ human serum. Cultureswere started at 0.5% and grown for 16 days with daily media changes.Beginning on day 14 exflagellation was monitored. On Day 16, allcultures were pooled and re divided into 4 flasks to which BKIs wereadded for 30 minutes at 50 nM, 100 nM, 300 nM, 1 μM and 3 μM with oneflask as a vehicle-only control and NA-PP2 flasks with 50 nM, 300 nM,and 3 μM. A wet mount was taken to check exflagellation and monitoredbeginning at 10 minutes and observed until 25 minutes had elapsed. Eachflask of culture was fed to approximately 150 4 day old A. stephensimosquitoes for 20 minutes. Ten days post feed ˜50 mosquitoes from eachcage was checked for midgut oocyst infection. On day 14 remainingmosquitoes in each cage were dissected and pooled to check for salivarygland sporozoites.

Administration of 50 mg/kg Compound 29 intraperitoneally (i.p.) resultedin a peak plasma concentration of 8.2 μM and plasma levels exceeding 0.4μM for 20 hours post-treatment (FIG. 17a ). When administered toinfected mice at this dose, Compound 29 completely suppressedexflagellation in blood samples from treated mice 30 minutespost-injection and up to 14 hours later (FIG. 17a ). However, 20 hoursafter dosing, when lower levels (0.41 μM) of Compound 29 could bedetected, some exflagellation was observed (FIG. 17a ), demonstratingthat the effect of Compound 29 is reversible. Compound 29 blockedexflagellation of P. berghei WT and P. berghei exogenously expressingPbCDPK4 or PfCDPK4 with similar IC₅₀s in vitro (FIG. 17b ).

P. berghei gametocyte-infected mice was injected i.p. with Compound 29at 10 mg/kg, a dose which has no impact on asexual parasitaemia orgametocyte rates, but is sufficient to block exflagellation (not shown)to assess its impact on malaria transmission to mosquitoes. Anophelesstephensi mosquitoes were allowed to feed on treated mice 30 minutesafter dosing. We used P. berghei with WT expression of PbCDPK4, becauseof the results of FIG. 17b , but expressing GFP for ease of followingoocyst production (FIG. 17c ). Treatment of mice at 10 mg/kg completelyblocked the formation of oocysts, while infected mice treated withvehicle or with control compound NA-PP2, gave rise to hundreds ofoocysts per mosquito (FIG. 17c ). Similarly, mixing Compound 29 (finalconcentrations of 1 μM or 3 μM) with human blood containing P.falciparum gametocytes, prevented the exflagellation of microgametes andthe infection of A. stephensi midguts, compared to vehicle control (FIG.17d ). Furthermore, a complete absence of infective sporozoites wasobserved in dissected salivary glands of A. stephensi fed with 3 μMCompound 29 treated blood (FIG. 17d ) which is consistent the absence ofexflagellation at this concentration. At 100 nM, Compound 29 stillreduced the prevalence of oocyst infections, and the sporozoite load inthe few salivary glands that were infected was diminished by 86%relative to untreated controls (FIG. 17d ). The observed overlap inphenotypic and developmental defects associated with CDPK4 inhibition inP. berghei and P. falciparum indicates that these enzymes have highlysimilar functional capacity. Compound 29 blocked exflagellation of P.falciparum microgametocytes and P. berghei microgametocytes expressingeither PbCDPK4 or PfCDPK4 (FIGS. 17b and 17d ) indicating that CDPK4 ofboth species can be blocked by the compound. Further, the active site ofP. vivax CDPK4 is highly homologous to that of PfCDPK4, so Compound 29and related compounds may be useful in blocking vivax malaria.

Biological Example 21 Inhibition of P. falciparum Proliferation

The following compounds were tested for inhibition P. falciparum(PfCDPK1, PfCDPK4) enzymes and ability to block P. falciparumproliferation according to the preceding methods:

TABLE 10 Inhibition of P. falciparum proliferation Enzyme Inhibition(EC50, μM) Proliferation PfCDPK1 PfCDPK4 Blocking Compound (% inhibition(% inhibition P. falciparum No. @ 3 uM) PfCDPK1 @ 3 uM) PfCDPK4 (EC₅₀,μM)  8 92 0.1559 92 0.091  9 76 88 10 93 0.3804 94 0.3282  1 51 25  21.25 0.48 40 11 93 0.3729 87 0.5486 12 95 0.6585 91 0.5256  3 78 76 >50  5f 15 13 25  5e 5  5a 52 14 13 15 11  5b 20  5c 45 23 16 13 17 44 5 184 19 14  5d 76 86  5g 56 75 20 0.309 93 0.2307  7a 97 86  7b 72 85  7c93 97  7d 90 80  7e 74 52   7f 96 85  7g 72 64  7h 83 85   7j 87 30   7m77 85   7i 79 79  7n 51 10  7o 58 79  7p 95 0.3805 98 0.1421  7z 79 21  7ab 85 79   7t 65 1.3569 83 0.1688   5i 58 80 0.6029  7q 0.20810.0414 >10   7aa 75 18   7r 94 1.2874 99 0.4288 >50   7s 53 20 21 68 922 71 9  5h 66 95 0.1543  7k 93 1.3614 43 >3  7u 94 0.4219 77 1.105   7l85 31 23 75 11  7v 27  7x 21   7w 55  7y 17 24 0.4709 93 0.3366 25 920.5785 26 93 0.5438 27 0.8794 44 >3   5j 48  5p 53   5m 42  5k 69 28  5q78 15  5n 49 27   5l 51 41   5r 55 22  5o 71 37 28 92 0.7394 99 0.018831.9 29 97 0.3527 101 0.0139 6.16 30 82 86 31 51 40 32 0.3652 96 0.055433 0.1959 98 0.0211 >10 34 0.0272 97 0.0302 5.83 35 0.1514 99 0.0312 >1036 0.1462 99 0.0157 11.7 37 0.0958 96 0.0716 38 0.0452 96 0.0349 7 39102 0.0211 98 0.0092 7 40 101 0.0963 84 0.5136 41 101 0.0363 980.0192 >10 42 101 0.1557 94 0.3084 43 100 0.2775 78 1.2037 44 100 0.181797 0.0583 45 101 0.0565 82 0.3497 46 95 0.4772 52 >3 47 91 1.2856 511.4322 48 84 2.279 10 >3 49 88 0.688 22 2.3458 50 83 2.0926 26 >3 51 861.5998 92 0.2595 52 93 1.0749 62 1.2272 39.5 53 98 0.5317 57 1.6358 54101 0.0478 77 0.4376 55 100 0.2160 94 0.1750 56 93 1.1375 73 0.8877 5785 0.7055 82 0.2427 58 97 0.2142 50 2.4330 59 98 0.1107 79 0.4586 60 980.0664 85 0.3257 >10 61 101 0.0714 80 0.5146 7 62 102 0.0377 950.1402 >10 63 103 0.0118 96 0.1355 >10 64 101 0.1279 66 1.0831 65 1010.1012 53 2.8532 66 100 0.2868 77 2.1832 67 96 0.3249 49 1.9874 68 702.2040 9 >3 69 101 0.1978 97 0.1320 81 73 75 82 56 >3 4 >3 70 100 0.053896 0.0834 71 101 0.0676 98 0.0652 72 94 0.9484 91 0.2412 73 87 14 74 970.3473 68 1.0132 75 75 5 83 96 1.0779 93 0.3714 84 101 0.0613 99 0.055185 99 0.2287 72 0.4788 90 77 64 91 73 59 92 68 51 93 64 49 94 71 51 9885 9 99 70 5 100  101 0.0749 68 0.9080 101  79 14 103  75 58 105  71 76107  65 52 109  54 30 111  63 41 113  65 61 115  71 84 117  71 74 118 97 0.5108 81 0.5116 119  102 0.0769 101 0.0073 120  101 0.0255 90 0.2135121  93 0.7017 93 0.1462 122  97 0.4817 32 >3 123  62 4 124  98 0.119393 0.1005 125  89 1.0892 93 0.1130 126  76 65 127  68 46 128  79 64 129 79 9 130  95 2.9475 47 >3 131  100 0.2659 38 >3 132  96 >3 96 1.4865133  79 26 134  89 87 135  96 0.6679 97 0.1374 136  90 16 137  95 2.432595 0.9418 139  99 1.1500 96 0.5658 140  98 0.3375 90 0.3988 141  921.4740 55 2.0305 142  98 0.2942 97 0.0947 143  97 97 144  95 94 145  9363 146  99 95 147  100 0.0668 99 0.0172 148  0.0390 149  0.0816 150 0.0337

Biological Example 22 Toxicity Data in Human Cell Lines

The following compounds were tested for toxicity in human cell linesaccording to the preceding methods:

TABLE 11 Toxicity data in human cell lines Cell Toxicity LD50 Results(μM) Observed Comp. HFF No. Toxicity HL60 CRL8155 SF539 HCC2998  1 <30%at 25 μM <30% at 25 μM <30% at 25 μM <30% at 25 μM  2 <30% at 25 μM; 37%at 25 μM; <30% at 25 μM <30% at 25 μM <30% at 30 μM <30% at 30 μM 11<30% at 10 μM <30% at 10 μM <30% at 10 μM <30% at 10 μM 12 <30% at 10 μM<30% at 10 μM <30% at 10 μM <30% at 10 μM  3 <30% at 10 μM <30% at 10 μM<30% at 10 μM <30% at 10 μM  5d <30% at 10 μM <30% at 10 μM <30% at 10μM <30% at 10 μM  5g <30% at 10 μM <30% at 10 μM <30% at 10 μM <30% at10 μM 20 <30% at 10 μM <30% at 10 μM  7c 51% at 25 μM 54% at 25 μM 31%at 25 μM <30% at 25 μM  7p 50% at 25 μM; 46% at 25 μM 84% at 25 μM; <30%at 25 μM 35% at 10 μM 54% at 10 μM   7t <30% at 10 μM <30% at 10 μM <30%at 10 μM <30% at 10 μM   5i <30% at 10 μM; <30% at 10 μM; <30% at 10 μM<30% at 10 μM <30% at 30 μM <30% at 30 μM  7q <30% at 10 μM <30% at 10μM <30% at 10 μM <30% at 10 μM   7r <30% at 10 μM <30% at 10 μM <30% at10 μM <30% at 10 μM  5h <30% at 10 μM <30% at 10 μM <30% at 10 μM <30%at 10 μM 25 47% toxicity at 38% toxicity at 10 μM 10 μM 26 45% toxicityat 38% toxicity at 10 μM 10 μM  5p <30% at 25 μM <30% at 25 μM <30% at25 μM <30% at 25 μM  5n <30% at 10 μM <30% at 10 μM <30% at 10 μM <30%at 10 μM 28 <30% at 10 μM <30% at 10 μM <30% at 10 μM <30% at 10 μM <30%at 30 μM <30% at 30 μM 29 <30% at 10 μM <30% at 10 μM <30% at 10 μM <30%at 10 μM 30 <30% at 10 μM <30% at 10 μM <30% at 10 μM <30% at 10 μM <30%at 30 μM <30% at 30 μM 33 T at 100 μM <30% at 10 μM <30% at 10 μM <30%at 10 μM 30% at 10 μM 34 T at 100 μM <30% at 10 μM <30% at 10 μM 6; 104%at 30% at 10 μM 10 μM 35 T at <30% at 10 μM <30% at 10 μM <30% at 10 μM<30% at 10 μM 25-100 μM 36 T at 100 μM <30% at 10 μM <30% at 10 μM <30%at 10 μM <30% at 10 μM 37 T at 100 μM 38 T at 100 μM <30% at 10 μM <30%at 10 μM 98% at 30 μM 98% at 30 μM 40 T at 100 μM 41 T at 100 μM <30% at10 μM <30% at 10 μM 42 T at 100 μM 44 T at 100 μM 46 <30% at 10 μM <30%at 10 μM 69 T at 25 μM <30% at 10 μM <30% at 10 μM 70 40% at 10 μM <30%at 10 μM 71 <30% at 10 μM <30% at 10 μM 72 <30% at 10 μM <30% at 10 μM83 37% at 10 μM <30% at 10 μM 38% at 30 μM 46% at 30 μM 84 <30% at 10 μM<30% at 10 μM 50% at 30 μM 48% at 30 μM 90 <30% at 10 μM <30% at 10 μM98 <30% at 10 μM <30% at 10 μM 105  <30% at 10 μM <30% at 10 μM 115 <30% at 10 μM <30% at 10 μM 118  T at 6.25 & 25 μM 119  <30% at 10 μM;<30% at 10 μM <30% at 30 μM <30% at 30 μM 120  T at 25 μM 121  <30% at10 μM <30% at 10 μM 122  <30% at 10 μM <30% at 10 μM <30% at 30 μM <30%at 30 μM 124  T at 6.25 & 25 μM 147  60% at 30 μM; <30% at 30 μM 33% at10 μM 148  49% at 30 μM; <30% at 30 μM <30% at 10 μM 149  <30% at 30 μM<30% at 10 μM <30% at 30 μM 150  T at 25 μM <30% at 10 μM <30% at 10 μM<30% at 30 μM <30% at 30 μM

Biological Example 23 Pharmacokinetics Properties

The following compounds were tested for their solubility properties inbuffers of various pH:

TABLE 12 Pharmacokinetics properties Experimental PK/PD Analysis ResultsOral Solubility (μM) Administration (IP Administration) Compound pH pHpH AUC (μM- No. 2.0 6.5 7.4 C_(max) (μM) T_(max) (min) min)  10 29 25.97150 6725.59  2 43 2.4 1.5  7a 45  7q 19.71 30 2120.14 (3.8 IP) (30 IP)(275.86 IP)  7r 12  28 49 58 56 4.41 160 1228.15 (84.06 IP) (40 IP)(9050.27 IP)  29 34 47 49 6.64 120 1200.96  34 23  35 45  36 0.59 220109.66  41 4.4 13.57 100 3172.19  42 4.4 10.69 160 2437.28  44 47 38 330.95 180 148.21  45 62  69 3  70 5  71 9  83 36  84 26 118 40 119 50 12044 121 43 0.19 90 26.69 124 41 1.84 180 399.24 125 54 132 3.8 134 19.3135 3.0 137 1.0 138 51 139 0.69 140 22 142 5.2 143 17 144 60 146 9 14779 148 56 149 75 150 82

Biological Example 24 Biological Properties of Compound Nos. 29 and 150

The compounds 29 and 150 were tested for their various biologicalproperties according to the preceeding methods:

TABLE 13 Biological properties of Compound Nos. 29 and 150 EnzymaticAssay Parasite Proliferation IC₅₀ Results Assay Com- (μM) EC₅₀ Results(μM) pound Tg Cp Pf T. C. P. falciparum No. CDPK1 CDPK1 CDPK4 gondiiparvum Exflaggelation  29 0.003 0.001 0.004 0.052 0.56 0.04 150 0.0030.001 0.010 0.137 0.54 0.05 Stability T_(1/2) (min) Mouse Oral (10 mg/kgOral (50 mg/kg Com- Liver Dose) Dose) pound Micro- C_(max) T_(max) AUCC_(max) T_(max) AUC No. somes (μM) (min) (μM-min) (μM) (min) (μM-min) 29 >60 0.2 140 57 1.1 160 311 150 >60 0.8 129 430 ND ND ND IP (10 mg/kgDose) IP (50 mg/kg Dose) Compound C_(max) T_(max) AUC C_(max) T_(max)AUC No. (μM) (min) (μM-min) (μM) (min) (μM-min)  29 0.7 30 191 8.2 301769 150 3 40 863 ND ND ND

Mice were dosed with 40 mg/kg of compound by oral gavages at 7 am, noon,and 3 pm on 4 consecutive days and blood levels determined by LC-MS. Thefirst and fourth troughs refer to compound levels 17 hours aftercompound dosing taken at the beginning of d2 and d5. The first peak was1 hr after the first dose. The fourth day peak was 1 hr after the thirddose of d4. Mean±SD of N=3:

Blood level (μM) Compound First Fourth D4 Fourth No. First Peak TroughPeak Trough 29 0.05 ± 0.08 0 ± 0 6.6 ± 1.6 0 ± 0 150 2.1 ± 1.2 2.0 ± 1.68.9 ± 3.4 6.3 ± 1.8

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CONCLUSION

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize.

All of the references cited herein are incorporated by reference.Aspects of the disclosure can be modified, if necessary, to employ thesystems, functions and concepts of the above references and applicationto provide yet further embodiments of the disclosure. These and otherchanges can be made to the disclosure in light of the detaileddescription.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure. Accordingly, the disclosure is not limited, except as by theappended claims.

We claim:
 1. A method for treating an apicomplexan protozoan relateddisease comprising providing to a patient in need of such treatment atherapeutically effective amount of a compound of formula:

or a pharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition comprising said compound and a pharmaceutically acceptableexcipient, carrier, or diluent, wherein X, Y, and Z are defined byeither: (i) X is N, Y is C, and Z is N; or (ii) X is C, Y is N, and Z isC(H); R¹ is C₂₋₆ alkyl, C₁₋₆ haloalkyl, —C₁₋₄ alkyl—R¹², C₂₋₆ alkenyl,C₂₋₆ alkynyl, C₃₋₈ cycloalkyl, monocyclic heterocyclyl, monocyclicheteroaryl, or phenyl, wherein the cycloalkyl, heterocyclyl, heteroaryl,and phenyl groups are each optionally substituted with one or two R¹¹groups; each R¹¹ is independently C₁₋₆ alkyl, C₁₋₆ haloalkyl, —C(O)R,—C(O)OR, —C(O)NR₂, —S(O)₂NR₂, or —S(O)₂R; and R¹² is —OR, —SR, —NR₂,—C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂,—N(R)C(O)R, —N(R)C(O)OR, —N(R)C(O)NR₂, phenyl, monocyclic heteroaryl,C₃₋₈ cycloalkyl, or monocyclic heterocyclyl, wherein the aryl,heteroaryl, C₃₋₈ cycloalkyl, and heterocyclyl groups are each optionallysubstituted by one, two, or three groups that are each independentlyhalogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR, —SR, —NR₂,—C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂,—N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂; R³ is one of the formulas,

wherein n is 0, 1, or 2; Q is —O—, —S—, or —N(R^(Q))—, wherein R^(Q) ishydrogen or C₁₋₆ alkyl; and R³³ is C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆haloalkyl, C₃₋₈ cycloalkyl, heterocyclyl, aryl, arylC₁₋₆ alkyl,heteroaryl, or heteroarylC₁₋₆ alkyl wherein the alkyl, aryl, arylalkyl,heteroaryl, and heteroarylalkyl are optionally substituted with one,two, three, or four groups that are each independently halogen, cyano,nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR²⁰, —SR²⁰, —N(R²⁰)₂, —C(O)R²⁰,—C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰, —OC(O)R²⁰, —OC(O)OR²⁰,—OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰, or —N(R²⁰)C(O)N(R²⁰)₂,wherein each R²⁰ is independently hydrogen or C₁₋₆ alkyl, each R³² isindependently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR³⁴,—SR³⁴, —N(R³⁴)₂, —C(O)R³⁴, —C(O)OR³⁴, —C(O)N(R³⁴)₂, —S(O)₂R³⁴,—OC(O)R³⁴, —OC(O)OR³⁴, —OC(O)N(R³⁴)₂, —N(R³⁴)C(O)R³⁴, —N(R³⁴)C(O)OR³⁴,or —N(R³⁴)C(O)N(R³⁴)₂ wherein each R³⁴ is independently hydrogen or C₁₋₆alkyl; and R³⁵ is hydrogen or C₁₋₆ alkyl; and each R is independentlyhydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆ haloalkyl, C₃₋₈ cycloalkyl,heterocyclyl, aryl, arylC₁₋₆ alkyl, heteroaryl, or heteroarylC₁₋₆ alkylwherein the alkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl areoptionally substituted with one, two, three, or four groups that areeach independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,—OR⁰, —SR⁰, —N(R⁰)₂, —C(O)R⁰, —C(O)OR⁰, —C(O)N(R⁰)₂, —S(O)₂R⁰, —OC(O)R⁰,—OC(O)OR⁰, —OC(O)N(R⁰)₂, —N(R⁰)C(O)R⁰, —N(R⁰C(O)OR⁰, or—N(R⁰)C(O)N(R⁰)₂, wherein each R⁰ is independently hydrogen or C₁₋₆alkyl, provided that the compound is not1-cyclopentyl-3-(1-methyl-1H-indol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine,1-cyclopentyl-3-(1H-indol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine,1-cyclopentyl-3-(1H-indol-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine,6-(4-amino-1-cyclopentyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indole-3-carbaldehyde,3-(1H-indol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine,1-isopropyl-3-(1-methyl-1H-indol-5-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine,3-(1H-indol-6-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine,2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-1H-indol-5-ol,3-(1H-indol-4-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine, and1-cyclopentyl-3-(1H-indol-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine. 2.The method of claim 1, wherein the apicomplexan protozoan relateddisease is toxoplasmosis, cryptosporidiosis, coccidiosis, or malaria. 3.The method of claim 1, wherein the compound is selected from:3-(6-isopropoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;1-(piperidin-4-ylmethyl)-3-(6-propoxynaphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(benzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-butoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(allyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(2-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(3-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(4-chlorobenzyloxy)naphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(benzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(allyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-butoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-isobutoxynaphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-isobutoxynaphthalen-2-yl)-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(2-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(3-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(2,5-dimethylbenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;1-isopropyl-3-(6-(2-methylbenzyloxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;1-isopropyl-3-(6-(2-methyl-5-(trifluoromethyl)benzyloxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(3-chloro-4-(2,2,2-trifluoroethyl)benzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(3-chloro-5-fluorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;1-isopropyl-3-(6-(1-phenylethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(4-tert-butylbenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;1-isopropyl-3-(6-(pyridin-4-ylmethoxy)naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;3-(6-(4-chlorobenzyloxy)naphthalen-2-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine;6-(4-amino-1-(piperidin-4-ylmethyl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-N,N-dimethylquinolin-2-amine;3-tert-butyl-1-(6-ethoxynaphthalen-2-yl)imidazo[1,5-a]pyrazin-8-amine;3-(6-ethoxynaphthalen-2-yl)-1-((l-methylpiperidin-4-yl)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine;and pharmaceutically acceptable salts thereof.
 4. The method of claim 1,wherein the compound is of the formula:


5. The method of claim 1, wherein the compound is of the formula:


6. The method of claim 1, wherein R³ is


7. The method of claim 6, wherein Q is —O— or —N(R^(Q))—.
 8. The methodof claim 7, wherein R³³ is C₁₋₆alkyl, C₂₋₆ alkenyl, arylC₁₋₆alkyl, orheteroarylC₁₋₆alkyl, wherein the arylalkyl and heteroarylalkyl areoptionally substituted with one, two, three, or four groups that areeach independently halogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl,—OR²⁰, δR²⁰ N(R²⁰)₂, —C(O)R²⁰, —C(O)OR²⁰, —C(O)N(R²⁰)₂, —S(O)₂R²⁰,—OC(O)R²⁰, —OC(O)OR²⁰, —OC(O)N(R²⁰)₂, —N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)OR²⁰,or —N(R²⁰)C(O)N(R²⁰)₂, wherein each R²⁰ is independently hydrogen orC₁₋₆ alkyl.
 9. The method of claim 1, wherein R¹ is C₂₋₆ alkyl or—C₁₋₄alkyl-R¹².
 10. The method of claim 9, wherein R¹ is —CH₂—R¹². 11.The method of claim 9, wherein R¹² is phenyl, monocyclic heteroaryl,C₃₋₈ cycloalkyl, or monocyclic heterocyclyl, wherein the phenyl,heteroaryl, C₃₋₈ cycloalkyl, and heterocyclyl groups are each optionallysubstituted by one, two, or three groups that are each independentlyhalogen, cyano, nitro, C₁₋₆ alkyl, C₁₋₆ haloalkyl, —OR, —SR, —NR₂,—C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂,—N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂.
 12. The method of claim 9,wherein R¹² is piperidinyl optionally substituted by one or two groupsthat are each independently C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂,—S(O)₂R, —OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or—N(R)C(O)NR₂.
 13. The method of claim 7, wherein R³³ is C₁₋₆ alkyl orC₃₋₈ cycloalkyl.
 14. The method of claim 13, wherein R¹ is —C₁₋₄alkyl-R¹².
 15. The method of claim 13, wherein R¹² is —OR or monocyclicheterocyclyl optionally substituted by one or two groups that are eachindependently halogen, C₁₋₆ alkyl, —C(O)R, —C(O)OR, —C(O)NR₂, —S(O)₂R,—OC(O)R, —OC(O)OR, —OC(O)NR₂, —N(R)C(O)R, —N(R)C(O)OR, or —N(R)C(O)NR₂.16. The method of claim 15, wherein R¹ is —C₄ alkyl-R¹² and R³³ is C₃₋₈cycloalkyl.
 17. The method of claim 16, wherein R¹² is —OR.